ALK7:ActRIIB heteromultimers and uses thereof

ABSTRACT

In certain aspects, the disclosure provides soluble heteromeric polypeptide complexes comprising an extracellular domain of an ALK7 receptor and an extracellular domain of ActRIIB In certain aspects, these ALK7:ActRIIB heteromultimers are can be used to improve metabolic parameters in a patient in need thereof. In certain aspects, these ALK7:ActRIIB heteromultimers are can be used to treat or prevent one or more kidney-associated disease or condition in a patient in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/251,935, filed Jan. 18, 2019 (now U.S. Pat. No. 11,028,145), which isa continuation of U.S. application Ser. No. 15/092,577, filed Apr. 6,2016 (now U.S. Pat. No. 10,227,392), which claims the benefit ofpriority to U.S. provisional application Ser. No. 62/143,579, filed Apr.6, 2015. The disclosures of the foregoing applications are herebyincorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 27, 2021, isnamed 1848179-0002-109-103_Seq.txt and is 148,450 bytes in size.

BACKGROUND OF THE INVENTION

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family is dividedinto two general phylogenetic clades: the more recently evolved membersof the superfamily, which includes TGF-betas, activins, and nodal andthe clade of more distantly related proteins of the superfamily, whichincludes a number of BMPs and GDFs [Hinck (2012) FEBS Letters586:1860-1870]. TGF-beta family members have diverse, oftencomplementary biological effects. By manipulating the activity of amember of the TGF-beta family, it is often possible to cause significantphysiological changes in an organism. For example, the Piedmontese andBelgian Blue cattle breeds carry a loss-of-function mutation in the GDF8(also called myostatin) gene that causes a marked increase in musclemass [Grobet et al. (1997) Nat Genet 17(1):71-4]. Furthermore, inhumans, inactive alleles of GDF8 are associated with increased musclemass and, reportedly, exceptional strength [Schuelke et al. (2004) NEngl J Med 350:2682-8].

Changes in fibrosis, muscle, bone, fat, red blood cells, and othertissues may be achieved by enhancing or inhibiting intracellularsignaling (e.g., SMAD 1, 2, 3, 5, and/or 8) that is mediated by ligandsof the TGF-beta family. Thus, there is a need for agents that regulatethe activity of various ligands of the TGF-beta superfamily.

SUMMARY OF THE INVENTION

As described herein, it has been discovered that an ALK7:ActRIIBheterodimer protein complex is a unique antagonist of ligands of theTGF-beta superfamily, exhibiting a different ligand-bindingprofile/selectivity compared to corresponding ActRIIB and ALK7homodimers. In particular, an exemplary ALK7:ActRIIB heterodimerdisplays enhanced binding to activin AC, activin C, and BMP5 compared toeither homodimer, retains strong binding to activin B as observed withActRIIB homodimer, and exhibits reduced binding to GDF11, GDF8, activinA, BMP10, BMP6, GDF3, and BMP9. In particular, ALK7:ActRIIB heterodimerdisplays low to no observable affinity for BMP9, whereas this ligandbinds strongly to ActRIIB homodimer. See FIG. 6 . These resultstherefore demonstrate that ALK7:ActRIIB heterodimers are a moreselective antagonists (inhibitors) of certain ligands of the TGF-betasuperfamily compared to ActRIIB homodimers. Accordingly, an ALK7:ActRIIBheterodimer will be more useful than an ActRIIB homodimer in certainapplications where such selective antagonism is advantageous. Examplesinclude therapeutic applications where it is desirable to antagonize oneor more of activin B, activin AC, activin C, and BMP5 with decreasedantagonism of one or more of activin A, BMP10, BMP6, GDF3, and BMP9.Moreover, an ALK7:ActRIIB heterodimer was shown to have therapeuticeffects in a mouse model of kidney disease as well as a beneficialcatabolic effects on adipose cells. Therefore, while not wishing to bebound to a particular mechanism of action, it is expected thatALK7:ActRIIB heteromultimers, as well as variants thereof, that bindto/inhibit at least one or more of activin (e.g., activin A, activin B,activin AB, activin C, activin E, activin AC, activin AE, activin BC andactivin BE), GDF8, GDF11, BMP10, BMP6, BMP5, GDF3, and/or nodal will beuseful agents for treating kidney disease, particularly certaindisorders of kidney disease such as inflammation and fibrosis, and/orpromoting beneficial catabolic effects on adipose tissue. Furthermore,it is expected that other antagonists (inhibitors), or combinations ofantagonists, that mimic the binding/inhibitory properties of theALK7:ActRIIB heterodimers as well as agents that directly or indirectlyantagonize ALK7 and/or ActRIIB receptors, agents that directly orindirectly antagonize ALK7 and/or ActRIIB-binding ligands, agents thatdirectly or indirectly antagonize downstream signaling mediator (e.g.,Smads), and/or agents that directly or indirectly antagonize TGFβsuperfamily co-receptors (e.g., Cripto or Cryptic) will have similarbiological effects. These antagonistic mimetic are collectively referredto herein as “ALK7:ActRIIB antagonists” or “ALK7:ActRIIB inhibitors”.

Therefore, the present disclosure provides, in part, heteromultimercomplexes (heteromultimers) comprising at least one ALK7 polypeptide andat least one ActRIIB polypeptide (ALK7:ActRIIB heteromultimers).Preferably, ALK7 polypeptides comprise a ligand-binding domain of anALK7 receptor, for example, a portion of the ALK7 extracellular domain.Similarly, ActRIIB polypeptides generally comprise a ligand-bindingdomain of an ActRIIB receptor, for example, a portion of the ActRIIBextracellular domain. Preferably, such ALK7 and ActRIIB polypeptides, aswell as resultant heteromultimers thereof, are soluble.

In certain aspects, an ALK7:ActRIIB heteromultimer comprises an ALK7amino acid sequence that is at least 70% identical to a polypeptide thatbegins at any one of amino acids 21-28 of SEQ ID NO: 9 (e.g., aminoacids 21, 22, 23, 24, 25, 26, 27, or 28) and ends at any one of aminoacids 92-113 of SEQ ID NO: 9 (e.g., amino acids 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,or 113) of SEQ ID NO: 9. For example, ALK7:ActRIIB heteromultimers maycomprise an amino acid sequence that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids 28-92 of SEQ ID NO: 9. In otherembodiments, ALK7:ActRIIB heteromultimers may comprise an ALK7 aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toamino acids 21-113 of SEQ ID NO: 9. In other embodiments, ALK7:ActRIIBheteromultimers may comprise an ALK7 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. In stillother embodiments, ALK7:ActRIIB heteromultimers may comprise an ALK7amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 20. In even other embodiments, ALK7:ActRIIBheteromultimers may comprise an ALK7 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39. In furtherembodiments, ALK7:ActRIIB heteromultimers may comprise an ALK7 aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 43. In further embodiments, ALK7:ActRIIB heteromultimers maycomprise an ALK7 amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 46.

In certain aspects, an ALK7:ActRIIB heteromultimer comprises an ActRIIBamino acid sequence that is at least 70% identical to a polypeptide thatbegins at any one of amino acids 20-29 of SEQ ID NO: 1 (e.g., 20, 21,22, 23, 24, 25, 26, 27, 28, or 29) and ends at any one of amino acids109-134 of SEQ ID NO: 1 (109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, or 134) of SEQ ID NO: 1. For example, ALK7:ActRIIBheteromultimers may comprise an amino acid sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to amino acids 29-109 of SEQ IDNO: 1. In further embodiments, ALK7:ActRIIB heteromultimers may comprisean ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 25-131. In other embodiments, ALK7:ActRIIBheteromultimers may comprise an ActRIIB amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In stillother embodiments, ALK7:ActRIIB heteromultimers may comprise an ActRIIBamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 3. In even other embodiments, ALK7:ActRIIB heteromultimersmay comprise an ActRIIB amino acid sequence that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 5. In still even otherembodiments, ALK7:ActRIIB heteromultimers may comprise an ActRIIB aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 6. In certain preferred embodiments, ALK7:ActRIIBheteromultimers do not comprise an ActRIIB polypeptide comprising anacidic amino acid (e.g., the naturally occurring amino acids E or D oran artificial acidic amino acid) at the position corresponding to L79 ofSEQ ID NO: 1.

Various combinations of the ALK7 and ActRIIB polypeptides describedherein are also contemplated with respect to ALK7:ActRIIBheteromultimers. For example, in certain aspects, an ALK7:ActRIIBheteromultimer may comprise, consist essentially of or consist of a) apolypeptide comprising, consisting essentially of, or consisting of anALK7 amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 28-92 of SEQ ID NO: 9; and b) a polypeptidecomprising, or consisting essentially of, or consisting of an ActRIIBamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto amino acids 29-109 of SEQ ID NO: 1. In further aspects, anALK7:ActRIIB heteromultimer may comprise, consist essentially of orconsist of a) a polypeptide comprising, consisting essentially of, orconsisting of an ALK7 amino acid sequence that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to amino acids 21-113 of SEQ ID NO: 9; andb) a polypeptide comprising, or consisting essentially of, or consistingof an ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids 25-131 of SEQ ID NO: 1. In other aspects,an ALK7:ActRIIB heteromultimer may comprise, consist essentially of orconsist of a) a polypeptide comprising, consisting essentially of, orconsisting of an ALK7 amino acid sequence that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 10; and b) a polypeptidecomprising, or consisting essentially of, or consisting of an ActRIIBamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 2. In other aspects, an ALK7:ActRIIB heteromultimer maycomprise, consist essentially of or consist of a) a polypeptidecomprising, consisting essentially of, or consisting of an ALK7 aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 20; and b) a polypeptide comprising, or consistingessentially of, or consisting of an ActRIIB amino acid sequence that isat least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. Inother aspects, an ALK7:ActRIIB heteromultimer may comprise, consistessentially of or consist of a) a polypeptide comprising, consistingessentially of, or consisting of an ALK7 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39; and b) apolypeptide comprising, or consisting essentially of, or consisting ofan ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 2. In other aspects, an ALK7:ActRIIBheteromultimer may comprise, consist essentially of or consist of a) apolypeptide comprising, consisting essentially of, or consisting of anALK7 amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 43; and b) a polypeptide comprising, orconsisting essentially of, or consisting of an ActRIIB amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO: 2. In other aspects, an ALK7:ActRIIB heteromultimer may comprise,consist essentially of or consist of a) a polypeptide comprising,consisting essentially of, or consisting of an ALK7 amino acid sequencethat is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46;and b) a polypeptide comprising, or consisting essentially of, orconsisting of an ActRIIB amino acid sequence that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 2. In even other aspects, anALK7:ActRIIB heteromultimer may comprise, consist essentially of orconsist of a) a polypeptide comprising, consisting essentially of, orconsisting of an ALK7 amino acid sequence that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 10; and b) a polypeptidecomprising, or consisting essentially of, or consisting of an ActRIIBamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 3. In even other aspects, an ALK7:ActRIIB heteromultimermay comprise, consist essentially of or consist of a) a polypeptidecomprising, consisting essentially of, or consisting of an ALK7 aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 20; and b) a polypeptide comprising, or consistingessentially of, or consisting of an ActRIIB amino acid sequence that isat least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. In evenother aspects, an ALK7:ActRIIB heteromultimer may comprise, consistessentially of or consist of a) a polypeptide comprising, consistingessentially of, or consisting of an ALK7 amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39; and b) apolypeptide comprising, or consisting essentially of, or consisting ofan ActRIIB amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 3. In even other aspects, an ALK7:ActRIIBheteromultimer may comprise, consist essentially of or consist of a) apolypeptide comprising, consisting essentially of, or consisting of anALK7 amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 43; and b) a polypeptide comprising, orconsisting essentially of, or consisting of an ActRIIB amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO: 3. In other aspects, an ALK7:ActRIIB heteromultimer may comprise,consist essentially of or consist of a) a polypeptide comprising,consisting essentially of, or consisting of an ALK7 amino acid sequencethat is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46;and b) a polypeptide comprising, or consisting essentially of, orconsisting of an ActRIIB amino acid sequence that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 3. In still other aspects, anALK7:ActRIIB heteromultimer may comprise, consist essentially of orconsist of a) a polypeptide comprising, consisting essentially of, orconsisting of an ALK7 amino acid sequence that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 10; and b) a polypeptidecomprising, or consisting essentially of, or consisting of an ActRIIBamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 5. In still even other aspects, an ALK7:ActRIIBheteromultimer may comprise, consist essentially of or consist of a) apolypeptide comprising, consisting essentially of, or consisting of anALK7 amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 10; and b) a polypeptide comprising, orconsisting essentially of, or consisting of an ActRIIB amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO: 6.

As described herein, ALK7:ActRIIB heteromultimer structures include, forexample, heterodimers, heterotrimers, heterotetramers, heteropentamers,and higher order heteromultimer complexes. See, e.g., FIGS. 1, 2, and8-10 . In certain preferred embodiments, ALK7:ActRIIB heteromultimersare heterodimers.

In certain aspects, ALK7 and/or ActRIIB polypeptides may be fusionproteins. For example, in some embodiments, an ALK7 polypeptide may be afusion protein comprising an ALK7 polypeptide domain and one or moreheterologous (non-ALK7) polypeptide domains. Similarly, in someembodiments, an ActRIIB polypeptide may be a fusion protein comprisingan ActRIIB polypeptide domain and one or more heterologous (non-ActRIIB)polypeptide domains. Optionally, ALK7 polypeptides are connecteddirectly (fused) to one or more heterologous domains, or an interveningsequence, such as a linker, may be positioned between the amino acidsequence of the ALK7 polypeptide and the one or more heterologousdomains. Similarly, the ActRIIB polypeptide may be connected directly(fused) to one or more heterologous domains, or an intervening sequence,such as a linker, may be positioned between the amino acid sequence ofthe ActRIIB polypeptide and the one or more heterologous domains.Linkers may correspond to the roughly 15 amino acid unstructured regionat the C-terminal end of the extracellular domain of ActRIIB or ALK7(the “tail”), or it may be an artificial sequence of between 5 and 15,20, 30, 50, 100 or more amino acids that are relatively free ofsecondary structure. A linker may be rich in glycine and prolineresidues and may, for example, contain repeating sequences ofthreonine/serine and glycines. Examples of linkers include, but are notlimited to, the sequences TGGG (SEQ ID NO: 17), SGGG (SEQ ID NO: 18),TGGGG (SEQ ID NO: 15), SGGGG (SEQ ID NO: 16), GGGGS (SEQ ID NO: 58),GGGG (SEQ ID NO: 14), and GGG (SEQ ID NO: 13). In some embodiments, theone or more heterologous domains that provide a desirable property tothe ALK7 and/or ActRIIB fusion proteins including, for example, improvedpharmacokinetics, easier purification, targeting to particular tissues,etc. For example, a heterologous domain of a fusion protein may enhanceone or more of in vivo stability, in vivo half-life,uptake/administration, tissue localization or distribution, formation ofprotein complexes, multimerization of the fusion protein, and/orpurification. An ALK7 or ActRIIB fusion protein may include animmunoglobulin Fc domain (wild-type or mutant) or a serum albumin. Insome embodiments, an ALK7 and/or ActRIIB polypeptides may comprise apurification subsequence, such as an epitope tag, a FLAG tag, apolyhistidine sequence, and a GST fusion.

In certain embodiments, ALK7:ActRIIB heteromultimers described hereincomprise an ALK7 polypeptide covalently, or non-covalently, associatedwith an ActRIIB polypeptide wherein the ALK7 polypeptide comprises anALK7 domain and an amino acid sequence of a first member (or secondmember) of an interaction pair and the ActRIIB polypeptide comprises anActRIIB polypeptide and an amino acid sequence of a second member (orfirst member) of the interaction pair. Interaction pairs describedherein are designed to promote dimerization or form higher ordermultimers. See, e.g., FIGS. 1, 2, and 8-10 . In some embodiments, theinteraction pair may be any two polypeptide sequences that interact toform a complex, particularly a heterodimeric complex although operativeembodiments may also employ an interaction pair that forms a homodimericsequence. The first and second members of the interaction pair may be anasymmetric pair, meaning that the members of the pair preferentiallyassociate with each other rather than self-associate (i.e., guidedinteraction pairs). Accordingly, first and second members of anasymmetric interaction pair may associate to form a heterodimericcomplex. Alternatively, the interaction pair may be unguided, meaningthat the members of the pair may associate with each other orself-associate without substantial preference and thus may have the sameor different amino acid sequences. Accordingly, first and second membersof an unguided interaction pair may associate to form a homodimercomplex or a heterodimeric complex. Optionally, the first member of theinteraction action pair (e.g., an asymmetric pair or an unguidedinteraction pair) associates covalently with the second member of theinteraction pair. Optionally, the first member of the interaction actionpair (e.g., an asymmetric pair or an unguided interaction pair)associates non-covalently with the second member of the interactionpair. Optionally, the first member of the interaction action pair (e.g.,an asymmetric pair or an unguided interaction pair) associates throughboth covalent and non-covalent mechanisms with the second member of theinteraction pair.

In some embodiments, ALK7 polypeptides are fusion proteins that comprisean Fc domain of an immunoglobulin. Similarly, in some embodiments,ActRIIB polypeptides are fusion proteins that comprise an Fc domain ofan immunoglobulin. Traditional Fc fusion proteins and antibodies areexamples of unguided interaction pairs, whereas a variety of engineeredFc domains have been designed as asymmetric interaction pairs [Spiess etal (2015) Molecular Immunology 67(2A): 95-106]. Therefore, a firstmember and/or a second member of an interaction pair described hereinmay comprise a constant domain of an immunoglobulin, including, forexample, the Fc portion of an immunoglobulin. For example, a firstmember of an interaction pair may comprise an amino acid sequence thatis derived from an Fc domain of an IgG (IgG1, IgG2, IgG3, or IgG4), IgA(IgA1 or IgA2), IgE, or IgM immunoglobulin. Such immunoglobulin domainsmay comprise one or more amino acid modifications (e.g., deletions,additions, and/or substitutions) that promote ALK7:ActRIIBheteromultimer formation. For example, the first member of aninteraction pair may comprise, consist essentially of, or consist of anamino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ IDNOs: 23-37. Similarly, a second member of an interaction pair maycomprise an amino acid sequence that is derived from an Fc domain of anIgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgA1 or IgA2), IgE, or IgM. Suchimmunoglobulin domains may comprise one or more amino acid modifications(e.g., deletions, additions, and/or substitutions) that promoteALK7:ActRIIB heteromultimer formation. For example, the second member ofan interaction pair may comprise, consist essentially of, or consist ofan amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one ofSEQ ID NOs: 23-37. In some embodiments, a first member and a secondmember of an interaction pair comprise Fc domains derived from the sameimmunoglobulin class and subtype. In other embodiments, a first memberand a second member of an interaction pair comprise Fc domains derivedfrom different immunoglobulin classes or subtypes.

In some embodiments, an ALK7:ActRIIB heterodimer comprises i) an ALK7polypeptide comprising, consisting essentially of, or consisting of anamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 76, and ii) an ActRIIB polypeptide comprising, consistingessentially of, or consisting of an amino acid sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73. In otherembodiments, an ALK7:ActRIIB heterodimer comprises i) an ALK7polypeptide comprising, consisting essentially of, or consisting of anamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 80, and ii) an ActRIIB polypeptide comprising, consistingessentially of, or consisting of an amino acid sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 78.

Optionally, a first member and/or a second member of an interaction pair(e.g., an asymmetric pair or an unguided interaction pair) comprise amodified constant domain of an immunoglobulin, including, for example, amodified Fc portion of an immunoglobulin. For example, protein complexesof the disclosure may comprise a first modified Fc portion of an IgGcomprising, consisting essentially of, or consisting of an amino acidsequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to an amino acid sequence selected from thegroup: SEQ ID NOs: 23-37 and a second modified Fc portion of an IgG,which may be the same or different from the amino acid sequence of thefirst modified Fc portion of the IgG, comprising, consisting essentiallyof, or consisting of an amino acid sequence that is at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anamino acid sequence selected from the group: SEQ ID NOs: 23-37. In someembodiments, ALK7:ActRIIB heteromultimers comprise, consist essentiallyof, or consist of: a) an ALK7 (or ActRIIB) fusion protein comprising animmunoglobulin domain that comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23, optionallywherein the immunoglobulin domain comprises a positively charged aminoacid (e.g., K, R, or H) at the positions corresponding to residues 134and 177 of SEQ ID NO: 23, and further optionally wherein theimmunoglobulin domain does not comprise a positively charged amino acid(e.g., K, R, or H) at the position corresponding to residue 225 of SEQID NO: 23, and b) an ActRIIB (or ALK7) fusion protein comprising animmunoglobulin domain that comprises, consisting essentially of, orconsisting of an amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 24, optionally wherein theimmunoglobulin domain comprises a negatively charged (e.g., D or E)amino acid at the positions corresponding to residues 170 and 187 of SEQID NO: 24, and further optionally wherein the immunoglobulin domaincomprises a positively charged amino acid (e.g., K, R, or H) at theposition corresponding to residue 225 of SEQ ID NO: 24. In otherembodiments, ALK7:ActRIIB heteromultimers comprise, consist essentiallyof, or consist of: a) an ALK7 (or ActRIIB) fusion protein comprising animmunoglobulin domain that comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27, optionallywherein the immunoglobulin domain comprises a C at the positioncorresponding to residue 132 of SEQ ID NO: 27 and a W at the positioncorresponding to residue 144 of SEQ ID NO: 27, and further optionallywherein the immunoglobulin domain does not comprise a positively chargedamino acid (e.g., K, R, or H) at the position corresponding to residue225 of SEQ ID NO: 27, and b) an ActRIIB (or ALK7) fusion proteincomprising an immunoglobulin domain that comprises, consistingessentially of, or consisting of an amino acid sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28, optionallywherein the immunoglobulin domain comprises a S at the positioncorresponding to residue 144 of SEQ ID NO: 28, an A at the positioncorresponding to residue 146 of SEQ ID NO: 28, and a V at the positioncorresponding to residue 185 of SEQ ID NO: 28, and further optionallywherein the immunoglobulin domain does not comprise a positively chargedamino acid (e.g., K, R, or H) at the position corresponding to residue225 of SEQ ID NO: 28.

Optionally, an ALK7 and/or ActRIIB polypeptide includes one or moremodified amino acid residues selected from: a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, and an amino acid conjugated to an organic derivatizing agent.ALK7 and/or ActRIIB polypeptides may comprise at least one N-linkedsugar, and may include two, three or more N-linked sugars. Suchpolypeptides may also comprise O-linked sugars. ALK7 and/or ActRIIBpolypeptides may be produced in a variety of cell lines that glycosylatethe protein in a manner that is suitable for patient use, includingengineered insect or yeast cells, and mammalian cells such as COS cells,CHO cells, HEK cells and NSO cells. In some embodiments an ALK7 and/orActRIIB polypeptide is glycosylated and has a glycosylation patternobtainable from a Chinese hamster ovary cell line. PreferablyALK7:ActRIIB heteromultimer complexes of the disclosure exhibit a serumhalf-life of at least 4, 6, 12, 24, 36, 48, or 72 hours in a mammal(e.g., a mouse or a human). Optionally, ALK7:ActRIIB heteromultimers mayexhibit a serum half-life of at least 6, 8, 10, 12, 14, 20, 25, or 30days in a mammal (e.g., a mouse or a human).

In certain aspects, ALK7:ActRIIB heteromultimers of the disclosure bindto one or more TGF-beta superfamily ligands. Optionally, ALK7:ActRIIBheteromultimers bind to one or more of these ligands with a K_(D) ofless than or equal to 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹²M. For example,in some embodiments, ALK7:ActRIIB heteromultimers bind to activin. Insome embodiments, ALK7:ActRIIB heteromultimers bind to activin A. Insome embodiments, ALK7:ActRIIB heteromultimers bind to activin B. Insome embodiments, ALK7:ActRIIB heteromultimers bind to activin C. Insome embodiments, ALK7:ActRIIB heteromultimers bind to activin AB. Insome embodiments, ALK7:ActRIIB heteromultimers bind to activin AC. Insome embodiments, ALK7:ActRIIB heteromultimers bind to activin BC. Insome embodiments, ALK7:ActRIIB heteromultimers bind to activin E. Insome embodiments, ALK7:ActRIIB heteromultimers bind to activin AE. Insome embodiments, ALK7:ActRIIB heteromultimers bind to activin BE. Insome embodiments, ALK7:ActRIIB heteromultimers bind to GDF11. In someembodiments, ALK7:ActRIIB heteromultimers bind to GDF8. In someembodiments, ALK7:ActRIIB heteromultimers bind to BMP6. In someembodiments, ALK7:ActRIIB heteromultimers bind to BMP10. In someembodiments, ALK7:ActRIIB heteromultimers bind to BMP5. In someembodiments, ALK7:ActRIIB heteromultimers bind to GDF3. In someembodiments, ALK7:ActRIIB heteromultimers bind to nodal. In someembodiments, ALK7:ActRIIB heteromultimers do not bind to or do notsubstantially bind to BMP9. In some embodiments, the ALK7:ActRIIBheteromultimer binds to BMP10 with weaker affinity compared to acorresponding ActRIIB homomultimer. In some embodiments, theALK7:ActRIIB heteromultimer binds to BMP9 with weaker affinity comparedto a corresponding ActRIIB homomultimer. In some embodiments, theALK7:ActRIIB heteromultimer binds to GDF3 with weaker affinity comparedto a corresponding ActRIIB homomultimer. In some embodiments, theALK7:ActRIIB heteromultimer binds to BMP6 with weaker affinity comparedto a corresponding ActRIIB homomultimer. In some embodiments, theALK7:ActRIIB heteromultimer binds to GDF8 with weaker affinity comparedto a corresponding ActRIIB homomultimer. In some embodiments, theALK7:ActRIIB heteromultimer binds to GDF11 with weaker affinity comparedto a corresponding ActRIIB homomultimer. In some embodiments, theALK7:ActRIIB heteromultimer binds to activin C with stronger affinitycompared to a corresponding ActRIIB homomultimer. In some embodiments,the ALK7:ActRIIB heteromultimer binds to activin AC with strongeraffinity compared to a corresponding ActRIIB homomultimer. In someembodiments, the ALK7:ActRIIB heteromultimer binds to BMP5 with strongeraffinity compared to a corresponding ActRIIB homomultimer.

In general, ALK7:ActRIIB heteromultimers of the disclosure antagonize(inhibit) one or more activities of at least one TGF-beta superfamilyligand, and such alterations in activity may be measured using variousassays known in the art, including, for example, a cell-based assay suchas those described herein. In certain aspects, ALK7:ActRIIBheteromultimers may be used to inhibit signaling (e.g., Smad 2/3 and/orSmad 1/5/8 signaling) mediated by one or more TGFβ superfamily ligandsin, for example, a cell-based assay. For example, in some embodiments,ALK7:ActRIIB heteromultimers inhibit activin signaling in a cell-basedassay. In some embodiments, ALK7:ActRIIB heteromultimers inhibit activinA signaling in a cell-based assay. In some embodiments, ALK7:ActRIIBheteromultimers inhibit activin B signaling in a cell-based assay. Insome embodiments, ALK7:ActRIIB heteromultimers inhibit activin Csignaling in a cell-based assay. In some embodiments, ALK7:ActRIIBheteromultimers inhibit activin AB signaling in a cell-based assay. Insome embodiments, ALK7:ActRIIB heteromultimers inhibit activin ACsignaling in a cell-based assay. In some embodiments, ALK7:ActRIIBheteromultimers inhibit activin BC signaling in a cell-based assay. Insome embodiments, ALK7:ActRIIB heteromultimers inhibit activin Esignaling in a cell-based assay. In some embodiments, ALK7:ActRIIBheteromultimers inhibit activin AE signaling in a cell-based assay. Insome embodiments, ALK7:ActRIIB heteromultimers inhibit activin BEsignaling in a cell-based assay. In some embodiments, ALK7:ActRIIBheteromultimers inhibit GDF11 signaling in a cell-based assay. In someembodiments, ALK7:ActRIIB heteromultimers inhibit GDF8 signaling in acell-based assay. In some embodiments, ALK7:ActRIIB heteromultimersinhibit BMP6 signaling in a cell-based assay. In some embodiments,ALK7:ActRIIB heteromultimers inhibit BMP10 signaling in a cell-basedassay. In some embodiments, ALK7:ActRIIB heteromultimers inhibit BMP5signaling in a cell-based assay. In some embodiments, ALK7:ActRIIBheteromultimers may inhibit GDF3 signaling in a cell-based assay. Insome embodiments, ALK7:ActRIIB heteromultimers may inhibit nodalsignaling in a cell-based assay. In some embodiments, ALK7:ActRIIBheteromultimers do not inhibit or do not substantially inhibit BMP9signaling in a cell-based assay. In some embodiments, ALK7:ActRIIBheteromultimers are weaker inhibitors of BMP9 signaling in a cell-basedassay compared to a corresponding ActRIIB homomultimer. In someembodiments, ALK7:ActRIIB heteromultimers are weaker inhibitors of BMP10signaling in a cell-based assay compared to a corresponding ActRIIBhomomultimer. In some embodiments, ALK7:ActRIIB heteromultimers areweaker inhibitors of BMP6 signaling in a cell-based assay compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK7:ActRIIBheteromultimers are weaker inhibitors of GDF3 signaling in a cell-basedassay compared to a corresponding ActRIIB homomultimer. In someembodiments, ALK7:ActRIIB heteromultimers are weaker inhibitors of GDF11signaling in a cell-based assay compared to a corresponding ActRIIBhomomultimer. In some embodiments, ALK7:ActRIIB heteromultimers areweaker inhibitors of GDF8 signaling in a cell-based assay compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK7:ActRIIBheteromultimers are weaker inhibitors of activin A signaling in acell-based assay compared to a corresponding ActRIIB homomultimer. Insome embodiments, ALK7:ActRIIB heteromultimers are stronger inhibitorsof activin C signaling in a cell-based assay compared to a correspondingActRIIB homomultimer. In some embodiments, ALK7:ActRIIB heteromultimersare stronger inhibitors of activin AC signaling in a cell-based assaycompared to a corresponding ActRIIB homomultimer. In some embodiments,ALK7:ActRIIB heteromultimers are stronger inhibitors of BMP5 signalingin a cell-based assay compared to a corresponding ActRIIB homomultimer.

Any of the ALK7:ActRIIB heteromultimers as well as ALK7:ActRIIBantagonists described herein may be formulated as a pharmaceuticalpreparation (compositions). In some embodiments, pharmaceuticalpreparations comprise a pharmaceutically acceptable carrier. Apharmaceutical preparation will preferably be pyrogen-free (meaningpyrogen free to the extent required by regulations governing the qualityof products for therapeutic use). A pharmaceutical preparation may alsoinclude one or more additional compounds such as a compound that is usedto treat a disorder/condition described herein. In general, ALK7:ActRIIBheteromultimer pharmaceutical preparations are substantially free ofALK7 and/or ActRIIB homomultimers. For example, in some embodiments,ALK7:ActRIIB heteromultimer pharmaceutical preparations comprise lessthan about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1%ALK7 homomultimers. In some embodiments, ALK7:ActRIIB heteromultimerpharmaceutical preparations comprise less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than about 1% ActRIIB homomultimers.

In certain aspects, the disclosure provides nucleic acids encoding anALK7 or ActRIIB polypeptide as described herein. For example, an ActRIIBnucleic acid may comprise, consists essentially of, or consists of anucleic acid that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe sequence of 73-396 of SEQ ID NO: 7 or one that hybridizes understringent conditions to the complement of nucleotides 73-396 of SEQ IDNO: 7. Such an nucleic acid may be one that comprises the sequence ofSEQ ID NOs: 8 or 72. In some embodiments, an ActRIIB nucleic acidscomprises, consists essentially of, or consists of a nucleotide sequencethat is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQID Nos: 7, 8, and 72. Similarly, an ALK7 nucleic acid may comprise,consists essentially of, or consists of a nucleic acid that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the sequence of 61-339 of SEQID NO: 11 or one that hybridizes under stringent conditions to thecomplement of nucleotides 61-339 of SEQ ID NO: 11. Such an ALK7 nucleicacid may be one that comprises the sequence of SEQ ID NOs: 12, 22, 41,45, or 75. In some embodiments, an ALK7 nucleic acids comprises,consists essentially of, or consists of a nucleotide sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 11,12, 22, 41, 45, or 75.

In certain aspects, the present disclosure provides nucleic acidssequence comprising a coding sequence for and ALK7 polypeptide and acoding sequence for the ActRIIB polypeptide. For example, in someembodiments, nucleic acids of the disclosure a) comprises, consistsessentially of, or consists of a nucleotide sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 11, 12,21, 22, 40, 41, 44, 45, or 75, and b) comprises, consists essentiallyof, or consists of a nucleotide sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to any one of SEQ ID Nos: 7, 8, or 72.Preferably, ALK7 and/or ActRIIB nucleic acids are isolated and/orrecombinant nucleic acids. Nucleic acids disclosed herein may beoperably linked to a promoter for expression. The present disclosurefurther provides vectors comprising such ALK7 and/or ActRIIBpolynucleotides as well as cells (e.g., CHO cells), preferably cellsisolated from a human or other vertebrate species, comprising such ALK7and/or ActRIIB polynucleotides as well as vectors comprising such ALK7and/or ActRIIB polynucleotides.

In certain aspects, ALK7 polypeptides and/or ActRIIB polypeptides may beexpressed in a mammalian cell line, optionally a cell line that mediatessuitably natural glycosylation of the ActRIIB or ALK7 protein so as todiminish the likelihood of an unfavorable immune response in a patient(including the possibility of veterinary patients). Human and CHO celllines have been used successfully, and it is expected that other commonmammalian expression vectors will be useful. Thus the disclosureprovides cultured cells comprising any of the nucleic acids disclosedherein. Such cells may be mammalian cells, including CHO cells, NSOcells, HEK cells and COS cells. Other cells may be chosen depending onthe species of the intended patient. Other cells are disclosed herein.Cultured cells are understood to mean cells maintained in laboratory orother man-made conditions (e.g., frozen, or in media) and not part of aliving organism.

In certain aspects, the disclosure provides methods for making any ofthe ALK7 and ActRIIB polypeptides described herein as well asALK7:ActRIIB heteromultimer complexes comprising such polypeptides. Sucha method may include expressing any of the nucleic acids disclosedherein in a suitable cell (e.g., CHO cell or a COS cell). For example,in some embodiments a method of making a heteromultimer comprising anALK7 polypeptide and an ActRIIB polypeptide comprises: a) culturing acell under conditions suitable for expression of an ALK7 polypeptide andan ActRIIB polypeptide, wherein the cell comprises an ALK7polynucleotide and an ActRIIB polynucleotide; and b) recovering theheteromultimer so expressed. Alternatively, a method of making aheteromultimer comprising an ALK7 polypeptide and an ActRIIB polypeptidemay comprise: a) culturing a first cell under conditions suitable forexpression of an ALK7 polypeptide, wherein the first cell comprises anALK7 polynucleotide; b) recovering the ALK7 polypeptide so expressed; c)culturing a second cell under conditions suitable for expression of anActRIIB polypeptide, wherein the second cell comprises an ActRIIBpolynucleotide; d) recovering the ActRIIB polypeptide so expressed; e)combining the recovered ALK7 polypeptide and the recovered ActRIIBpolypeptide under conditions suitable for ALK7:ActRIIB heteromultimerformation; and f) recovering the ALK7:ActRIIB heteromultimer. In certainembodiments, ALK7 and/or ActRIIB polypeptides are expressed using a TPAleader sequence (e.g., SEQ ID NO: 70). In certain embodiments, ALK7and/or ActRIIB polypeptides are expressed in a CHO cell. ALK7 andActRIIB polypeptides described herein, as well as protein complexes ofthe same, may be recovered as crude, partially purified, or highlypurified fractions using any of the well-known techniques for obtainingprotein from cell cultures. In general, such methods result inALK7:ActRIIB complexes that are substantially free of ALK7 and/orActRIIB homomultimers. For example, in some embodiments, methods forproducing ALK7:ActRIIB heteromultimers result in less than about 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% ALK7homomultimers. In some embodiments, methods for producing ALK7:ActRIIBheteromultimers result in less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, or less than about 1% ActRIIB homomultimers. In someembodiments, methods for producing ALK7:ActRIIB heteromultimers resultin less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less thanabout 1% ALK7 homomultimers and In some embodiments, methods forproducing ALK7:ActRIIB heteromultimers result in less than about 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% ActRIIBhomomultimers.

The disclosure further provides methods and ALK7:ActRIIB heteromultimersfor use in the treatment or prevention of various activin C-associateddiseases and conditions. For example, in some embodiments, ALK7:ActRIIBheteromultimers may be used to increase fertility, increase spermproduction, increase seminiferous tubule volume, decrease prostateinflammation, decrease liver inflammation in a patient in need thereof.In some embodiments, ALK7:ActRIIB heteromultimers may be used to treator prevent hepatic cancer, testicular cancer, or prostate cancer. Insome embodiments, ALK7:ActRIIB heteromultimers may be used to decreaselevels (e.g., serum levels) of activin C in a patient in need thereof.

The disclosure further provides methods and ALK7:ActRIIB antagonists(e.g., ALK7:ActRIIB heteromultimers) for use in the treatment orprevention of various ALK7:ActRIIB-associated diseases and conditionsassociated with, for example, kidney, fat, fibrosis, and other tissues.Such diseases or conditions include, for example, chronic kidney diseaseor failure, acute kidney disease or failure, patients that have stage 1kidney disease, patients that have stage 2 kidney disease, patients thathave stage 3 kidney disease, patients that have stage 4 kidney disease,patients that have stage 5 kidney disease, non-diabetic kidney diseases,glomerulonephritis, interstitial nephritis, diabetic kidney diseases,diabetic nephropathy, glomerulosclerosis, rapid progressiveglomerulonephritis, renal fibrosis, Alport syndrome, IDDM nephritis,mesangial proliferative glomerulonephritis, membranoproliferativeglomerulonephritis, crescentic glomerulonephritis, renal interstitialfibrosis, focal segmental glomerulosclerosis, membranous nephropathy,minimal change disease, pauci-immune rapid progressiveglomerulonephritis, IgA nephropathy, polycystic kidney disease, Dent'sdisease, nephrocytinosis, Heymann nephritis, autosomal dominant (adult)polycystic kidney disease, autosomal recessive (childhood) polycystickidney disease, acute kidney injury, nephrotic syndrome, renal ischemia,podocyte diseases or disorders, proteinuria, glomerular diseases,membranous glomerulonephritis, focal segmental glomerulonephritis,pre-eclampsia, eclampsia, kidney lesions, collagen vascular diseases,benign orthostatic (postural) proteinuria, IgM nephropathy, membranousnephropathy, sarcoidosis, diabetes mellitus, kidney damage due to drugs,Fabry's disease, aminoaciduria, Fanconi syndrome, hypertensivenephrosclerosis, interstitial nephritis, Sickle cell disease,hemoglobinuria, myoglobinuria, Wegener's Granulomatosis, GlycogenStorage Disease Type 1, chronic kidney disease, chronic renal failure,low Glomerular Filtration Rate (GFR), nephroangiosclerosis, lupusnephritis, ANCA-positive pauci-immune crescentic glomerulonephritis,chronic allograft nephropathy, nephrotoxicity, renal toxicity, kidneynecrosis, kidney damage, glomerular and tubular injury, kidneydysfunction, nephritic syndrome, acute renal failure, chronic renalfailure, proximal tubal dysfunction, acute kidney transplant rejection,chronic kidney transplant rejection, non-IgA mesangioproliferativeglomerulonephritis, postinfectious glomerulonephritis, vasculitides withrenal involvement of any kind, any hereditary renal disease, anyinterstitial nephritis, renal transplant failure, kidney cancer, kidneydisease associated with other conditions (e.g., hypertension, diabetes,and autoimmune disease), Dent's disease, nephrocytinosis, Heymannnephritis, a primary kidney disease, a collapsing glomerulopathy, adense deposit disease, a cryoglobulinemia-associated glomerulonephritis,an Henoch-Schonlein disease, a postinfectious glomerulonephritis, abacterial endocarditis, a microscopic polyangitis, a Churg-Strausssyndrome, an anti-GBM-antibody mediated glomerulonephritis, amyloidosis,a monoclonal immunoglobulin deposition disease, a fibrillaryglomerulonephritis, an immunotactoid glomerulopathy, ischemic tubularinjury, a medication-induced tubulo-interstitial nephritis, a toxictubulo-interstitial nephritis, an infectious tubulo-interstitialnephritis, a bacterial pyelonephritis, a viral infectioustubulo-interstitial nephritis which results from a polyomavirusinfection or an HIV infection, a metabolic-induced tubulo-interstitialdisease, a mixed connective disease, a cast nephropathy, a crystalnephropathy which may results from urate or oxalate or drug-inducedcrystal deposition, an acute cellular tubulo-interstitial allograftrejection, a tumoral infiltrative disease which results from a lymphomaor a post-transplant lymphoproliferative disease, an obstructive diseaseof the kidney, vascular disease, a thrombotic microangiopathy, anephroangiosclerosis, an atheroembolic disease, a mixed connectivetissue disease, a polyarteritis nodosa, a calcineurin-inhibitorinduced-vascular disease, an acute cellular vascular allograftrejection, an acute humoral allograft rejection, early renal functiondecline (ERFD), end stage renal disease (ESRD), renal vein thrombosis,acute tubular necrosis, renal occlusion, acute interstitial nephritis,established chronic kidney disease, renal artery stenosis, ischemicnephropathy, uremia, drug and toxin-induced chronic tubulointerstitialnephritis, reflux nephropathy, kidney stones, Goodpasture's syndrome,normocytic normochromic anemia, renal anemia, diabetic chronic kidneydisease, IgG4-related disease, von Hippel-Lindau syndrome, tuberoussclerosis, nephronophthisis, medullary cystic kidney disease, renal cellcarcinoma, adenocarcinoma, nephroblastoma, lymphoma, leukemia,hyposialylation disorder, chronic cyclosporine nephropathy, renalreperfusion injury, renal dysplasia, azotemia, bilateral arterialocclusion, acute uric acid nephropathy, hypovolemia, acute bilateralobstructive uropathy, hypercalcemic nephropathy, hemolytic uremicsyndrome, acute urinary retention, malignant nephrosclerosis, postpartumglomerulosclerosis, scleroderma, non-Goodpasture's anti-GBM disease,microscopic polyarteritis nodosa, allergic granulomatosis, acuteradiation nephritis, post-streptococcal glomerulonephritis,Waldenstrom's macroglobulinemia, analgesic nephropathy, arteriovenousfistula, arteriovenous graft, dialysis, ectopic kidney, medullary spongekidney, renal osteodystrophy, solitary kidney, hydronephrosis,microalbuminuria, uremia, haematuria, hyperlipidemia, hypoalbuminaemia,lipiduria, acidosis, and hyperkalemia. In some embodiments, thedisclosure further provides methods and ALK7:ActRIIB antagonists (e.g.,ALK7:ActRIIB heteromultimers) for use in delaying or preventingprogression from: stage 1 to stage 2 kidney disease, stage 2 to stage 3kidney disease, stage 3 to stage 4 kidney disease, or stage 4 to stage 5kidney disease. In some embodiments, the disclosure further providesmethods and ALK7:ActRIIB antagonists (e.g., ALK7:ActRIIBheteromultimers) for use in preventing or reducing kidney inflammation.In some embodiments, the disclosure further provides methods andALK7:ActRIIB antagonists (e.g., ALK7:ActRIIB heteromultimers) for use inpreventing or reducing kidney damage. In some embodiments, thedisclosure further provides methods and ALK7:ActRIIB antagonists (e.g.,ALK7:ActRIIB heteromultimers) for use in preventing or reducing kidneyfibrosis.

In some embodiments, the disclosure further provides methods andALK7:ActRIIB antagonists (e.g., ALK7:ActRIIB heteromultimers) for use inlipolysis activity in a subject in need thereof. In some embodiments,the disclosure further provides methods and ALK7:ActRIIB antagonists(e.g., ALK7:ActRIIB heteromultimers) for use in decreasing body fatcontent or reducing the rate of increase in body fat content in asubject in need thereof. In some embodiments, the disclosure furtherprovides methods and ALK7:ActRIIB antagonists (e.g., ALK7:ActRIIBheteromultimers) for use in treating a disorder or condition associatedwith undesirable body weight gain in a subject. Such disorders include,for example, obesity (e.g., abdominal obesity); overweight; insulinresistance; metabolic syndrome and other metabolic diseases orconditions; a lipid disorder such as, low HDL levels, high LDL levels,hyperlipidemia, hypertriglyceridemia or dyslipidemia; lipoproteinaberrations; decreased triglycerides; fatty liver disease; non-alcoholicfatty liver disease; hyperglycemia; impaired glucose tolerance (IGT);hyperinsulinemia; high cholesterol (e.g., high LDL levels and/orhypercholesterolemia); cardiovascular disease such as, heart diseaseincluding coronary heart disease, congestive heart failure,atherosclerosis; arteriosclerosis, and/or hypertension; Syndrome X;vascular restenosis; neuropathy; and/or other disorders/conditionsassociated with one or more of the above diseases or conditions, and/orwith overweight (e.g., BMI of ≥25 kg/m²), or with too much body fat. Insome embodiments, the disclosure provides methods for decreasing bodyfat content or reducing the rate of increase in body fat content in asubject, comprising administering to a subject in need thereof aneffective amount of an ALK7:ActRIIB antagonist, or combination ofALK7:ActRIIB antagonists. In some embodiments, the disclosure providesmethods for treating a disorder associated with undesirable body weightgain in a subject, comprising administering to a subject in need thereofan effective amount of an ALK7:ActRIIB antagonist, or combination ofALK7:ActRIIB antagonists. In some embodiments, the disorder associatedwith undesirable body weight gain in a subject is selected from thegroup consisting of: obesity, non-insulin dependent diabetes mellitus(NIDDM), cardiovascular disease, hypertension, osteoarthritis, stroke,respiratory problems, and gall bladder disease. In some embodiments, thedisclosure provides methods for reducing cholesterol and/ortriglycerides in a patient, comprising administering to a subjectpatient in need thereof an effective amount of an ALK7:ActRIIBantagonist, or combination of ALK7:ActRIIB antagonists.

In some embodiments, the disclosure further provides methods andALK7:ActRIIB antagonists (e.g., ALK7:ActRIIB heteromultimers) for use intreating and/or ameliorating cancer or a condition associated withcancer. In some embodiments, the subject has a cancer selected from thegroup consisting of melanoma, breast, colon, and endometrial,pancreatic, gastric, and uterine cancer. In some embodiments, thesubject has myeloma (e.g., multiple myeloma, plasmacytoma, localizedmyeloma, and extramedullary myeloma). In some embodiments, theALK7:ActRIIB antagonist is administered to treat or prevent lymphaticmetastasis, bloodstream metastasis, tumor growth, or tumor invasion.

In some embodiments, the disclosure further provides methods andALK7:ActRIIB antagonists (e.g., ALK7:ActRIIB heteromultimers) for use intreating or preventing fibrosis or a disorder or condition associatedwith fibrosis in a patient in need thereof. Such disorders or conditionsinclude, for example, pulmonary fibrosis, hypersensitivity pneumonitis,idiopathic fibrosis, tuberculosis, pneumonia, cystic fibrosis, asthma,chronic obstructive pulmonary disease (COPD), emphysema, renal (kidney)fibrosis, renal (kidney) failure, chronic renal (kidney) disease, bonefibrosis, myelofibrosis, rheumatoid arthritis, systemic lupuserythematosus, scleroderma, sarcoidosis, granulomatosis withpolyangiitis, Peyronie's disease, liver fibrosis, Wilson's disease,glycogen storage diseases (particularly types III, IV, IX, and X),iron-overload, Gaucher disease, Zellweger syndrome, nonalcoholic andalcoholic steatohepatitis, biliary cirrhosis, sclerosing cholangitis,Budd-Chiari syndrome, surgery-associated fibrosis, Crohn's disease,Duputren's contracture, mediastinal fibrosis, nephrogeneic fibrosis,retroperitoneal fibrosis, atrial fibrosis, endomyocardial fibrosis,pancreatic fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show two schematic examples of heteromeric proteincomplexes comprising type I receptor and type II receptor polypeptides.FIG. 1A depicts a heterodimeric protein complex comprising one type Ireceptor fusion polypeptide and one type II receptor fusion polypeptide,which can be assembled covalently or noncovalently via a multimerizationdomain contained within each polypeptide chain. Two assembledmultimerization domains constitute an interaction pair, which can beeither guided or unguided. FIG. 1B depicts a heterotetrameric proteincomplex comprising two heterodimeric complexes as depicted in FIG. 1A.Complexes of higher order can be envisioned.

FIG. 2 show a schematic example of a heteromeric protein complexcomprising a type I receptor polypeptide (indicated as “I”) (e.g. apolypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain ofan ALK7 protein from humans or other species such as those describedherein, e.g., SEQ ID Nos: 9, 10, 19, 20, 38, 39, 42, 43, 46, 74, 76, 79,and 80) and a type II receptor polypeptide (indicated as “II”) (e.g. apolypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain ofan ActRIIB protein from humans or other species as such as thosedescribed herein, e.g., SEQ ID Nos: 1, 2, 3, 4, 5, 6, 71, 73, 77, and78). In the illustrated embodiments, the type I receptor polypeptide ispart of a fusion polypeptide that comprises a first member of aninteraction pair (“C₁”), and the type II receptor polypeptide is part ofa fusion polypeptide that comprises a second member of an interactionpair (“C₂”). In each fusion polypeptide, a linker may be positionedbetween the type I or type II receptor polypeptide and the correspondingmember of the interaction pair. The first and second members of theinteraction pair may be a guided (asymmetric) pair, meaning that themembers of the pair associate preferentially with each other rather thanself-associate, or the interaction pair may be unguided, meaning thatthe members of the pair may associate with each other or self-associatewithout substantial preference and may have the same or different aminoacid sequences. Traditional Fc fusion proteins and antibodies areexamples of unguided interaction pairs, whereas a variety of engineeredFc domains have been designed as guided (asymmetric) interaction pairs[e.g., Spiess et al (2015) Molecular Immunology 67(2A): 95-106].

FIG. 3 shows an alignment of extracellular domains of human ActRIIA (SEQID NO: 49) and human ActRIIB (SEQ ID NO: 2) with the residues that arededuced herein, based on composite analysis of multiple ActRIIB andActRIIA crystal structures, to directly contact ligand indicated withboxes.

FIG. 4 shows a multiple sequence alignment of various vertebrate ActRIIBprecursor proteins without their intracellular domains (SEQ ID NOs:50-55) human ActRIIA precursor protein without its intracellular domain(SEQ ID NO: 56), and a consensus ActRII precursor protein (SEQ ID NO:57).

FIG. 5 shows multiple sequence alignment of Fc domains from human IgGisotypes using Clustal 2.1 (SEQ ID NOs: 31, 35 and 32-33, respectively,in order of appearance). Hinge regions are indicated by dottedunderline. Double underline indicates examples of positions engineeredin IgG1 Fc to promote asymmetric chain pairing and the correspondingpositions with respect to other isotypes IgG2, IgG3 and IgG4.

FIG. 6 shows comparative ligand binding data for an ALK7-Fc:ActRIIB-Fcheterodimeric protein complex compared to ActRIIB-Fc homodimer andALK7-Fc homodimer. For each protein complex, ligands are ranked byk_(off), a kinetic constant that correlates well with ligand signalinginhibition, and listed in descending order of binding affinity (ligandsbound most tightly are listed at the top). At left, yellow, red, green,and blue lines indicate magnitude of the off-rate constant. Solid blacklines indicate ligands whose binding to heterodimer is enhanced orunchanged compared with homodimer, whereas dashed red lines indicatesubstantially reduced binding compared with homodimer. As shown, four ofthe five ligands with strong binding to ActRIIB-Fc homodimer (activin A,BMP10, GDF8, and GDF11) exhibit reduced binding to theActRIIB-Fc:ALK7-Fc heterodimer, the exception being activin B whichretains tight binding to the heterodimer. Similarly, three of fourligands with intermediate binding to ActRIIB-Fc homodimer (GDF3, BMP6,and particularly BMP9) exhibit reduced binding to the ActRIIB-Fc:ALK7-Fcheterodimer, whereas binding to activin AC is increased to become thesecond strongest ligand interaction with the heterodimer overall.Finally, activin C and BMP5 unexpectedly bind the ActRIIB-Fc:ALK7heterodimer with intermediate strength despite no binding (activin C) orweak binding (BMP5) to ActRIIB-Fc homodimer. No ligands tested bind toALK7-Fc homodimer.

FIG. 7 shows a multiple sequence alignment of ALK7 extracellular domainsderived from various vertebrate species (SEQ ID NOs: 59-64).

FIGS. 8A-8D show schematic examples of heteromeric protein complexescomprising an ALK7 polypeptide (e.g. a polypeptide that is at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100%identical to an extracellular domain of an ALK7 protein from humans orother species such as those described herein, e.g., SEQ ID Nos: 9, 10,19, 20, 38, 39, 42, 43, 46, 74, 76, 79, and 80) and an ActRIIBpolypeptide (e.g. a polypeptide that is at least 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to anextracellular domain of an ActRIIB protein from humans or other speciessuch as those described herein, e.g., SEQ ID Nos: 1, 2, 3, 4, 5, 6, 71,73, 77, and 78). In the illustrated embodiments, the ALK7 polypeptide ispart of a fusion polypeptide that comprises a first member of aninteraction pair (“C₁”), and the ActRIIB polypeptide is part of a fusionpolypeptide that comprises a second member of an interaction pair(“C₂”). Suitable interaction pairs included, for example, heavy chainand/or light chain immunoglobulin interaction pairs, truncations, andvariants thereof such as those described herein [e.g., Spiess et al(2015) Molecular Immunology 67(2A): 95-106]. In each fusion polypeptide,a linker may be positioned between the ALK7 or ActRIIB polypeptide andthe corresponding member of the interaction pair. The first and secondmembers of the interaction pair may be unguided, meaning that themembers of the pair may associate with each other or self-associatewithout substantial preference, and they may have the same or differentamino acid sequences. See FIG. 8A. Alternatively, the interaction pairmay be a guided (asymmetric) pair, meaning that the members of the pairassociate preferentially with each other rather than self-associate. SeeFIG. 8B. Complexes of higher order can be envisioned. See FIGS. 8C and8D.

FIGS. 9A-9G show schematic examples of heteromeric protein complexescomprising two ALK7 polypeptides (e.g. polypeptide that areindependently at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,97%, 98%, 99% or 100% identical to an extracellular domain of an ALK7protein from humans or other species such as those described herein,e.g., SEQ ID Nos: 9, 10, 19, 20, 38, 39, 42, 43, 46, 74, 76, 79, and 80)and two ActRIIB polypeptides (e.g. two polypeptides that areindependently at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,97%, 98%, 99% or 100% identical to an extracellular domain of an ActRIIBprotein from humans or other species such as those described herein,e.g., SEQ ID Nos: 1, 2, 3, 4, 5, 6, 71, 73, 77, and 78).

In the illustrated embodiment 9A, the first ALK7 polypeptide (from leftto right) is part of a fusion polypeptide that comprises a first memberof an interaction pair (“C₁”) and further comprises an additional firstmember of an interaction pair (“A₁”); and the second ALK7 polypeptide ispart of a fusion polypeptide that comprises a second member of aninteraction pair (“C₂”) and further comprises a first member of aninteraction pair (“A₂”). The first ActRIIB polypeptide (from left toright) is part of a fusion polypeptide that comprises a second member ofan interaction pair (“B₁”); and the second ActRIIB polypeptide is partof a fusion polypeptide that comprises a second member of an interactionpair (“B₂”). A₁ and A₂ may be the same or different; B₁ and B₂ may bethe same or different, and C₁ and C₂ may be the same or different. Ineach fusion polypeptide, a linker may be positioned between the ALK7 orActRIIB polypeptide and the corresponding member of the interaction pairas well as between interaction pairs. FIG. 9A is an example of anassociation of unguided interaction pairs, meaning that the members ofthe pair may associate with each other or self-associate withoutsubstantial preference and may have the same or different amino acidsequences.

In the illustrated embodiment 9B, the first ActRIIB polypeptide (fromleft to right) is part of a fusion polypeptide that comprises a firstmember of an interaction pair (“C₁”) and further comprises an additionalfirst member of an interaction pair (“A₁”); and the second ActRIIBpolypeptide is part of a fusion polypeptide that comprises a secondmember of an interaction pair (“B₂”). The first ALK7 polypeptide (fromleft to right) is part of a fusion polypeptide that comprises a secondmember of an interaction pair (“B₁”); and the second ALK7 polypeptide ispart of a fusion polypeptide that comprises a second member of aninteraction pair (“C₂”) and further comprises a first member of aninteraction pair (“A₂”). In each fusion polypeptide, a linker may bepositioned between the ALK7 or ActRIIB polypeptide and the correspondingmember of the interaction pair as well as between interaction pairs.FIG. 9B is an example of an association of guided (asymmetric)interaction pairs, meaning that the members of the pair associatepreferentially with each other rather than self-associate.

Suitable interaction pairs included, for example, heavy chain and/orlight chain immunoglobulin interaction pairs, truncations, and variantsthereof as described herein [e.g., Spiess et al (2015) MolecularImmunology 67(2A): 95-106]. Complexes of higher order can be envisioned.See FIG. 9C-9F. Using similar methods, particularly those that employlight and/or heavy chain immunoglobulins, truncations, or variantsthereof, interaction pairs may be used to produce ALK7:ActRIIBheterodimers that resemble antibody Fab and F(ab′)₂ complexes [e.g.,Spiess et al (2015) Molecular Immunology 67(2A): 95-106]. See FIG. 9G.

FIGS. 10A and 10B show schematic examples of a heteromeric proteincomplex comprising an ALK7 polypeptide (e.g. a polypeptide that is atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or100% identical to an extracellular domain of an ALK7 protein from humansor other species as described herein, e.g., SEQ ID Nos: 9, 10, 19, 20,38, 39, 42, 43, 46, 74, 76, 79, and 80), an ActRIIB polypeptide (e.g. apolypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 97%, 98%, 99% or 100% identical to an extracellular domain ofan ActRIIB protein from humans or other species as such as thosedescribed herein, e.g., SEQ ID Nos: 1, 2, 3, 4, 5, 6, 71, 73, 77, and78), and a ligand-binding domain of an antibody (e.g., a ligand bindingdomain derived from an antibody that binds to one or moreALK7:ActRIIB-binding ligands). In the illustrated embodiments, the ALK7polypeptide is part of a fusion polypeptide that comprises a firstmember of an interaction pair (“C₁”), and further comprises anadditional first member of an interaction pair (“A₁”). The ActRIIBpolypeptide is part of a fusion polypeptide that comprises a secondmember of an interaction pair (“B₁”). The variable heavy chain (V_(H))polypeptide is part of a fusion polypeptide that comprises a secondmember of an interaction pair (“C₂”), and further comprises a firstmember of an interaction pair (“A₂”). The variable heavy chain (V_(L))polypeptide is part of a fusion polypeptide that comprises a secondmember of an interaction pair (“B₂”). In each fusion polypeptide, alinker may be positioned between the ALK7 or ActRIIB polypeptide and thecorresponding member of the interaction pair, between interaction pairs,and between the V_(H) and V_(L) polypeptides and a member of theinteraction pair. A₁ and A₂ may be the same or different; B₁ and B₂ maybe the same or different, and C₁ and C₂ may be the same or different.Suitable interaction pairs included, for example, constant heavy chainand/or light chain immunoglobulin interaction pairs, truncations, andvariants thereof as described herein [e.g., Spiess et al (2015)Molecular Immunology 67(2A): 95-106]. FIG. 10A is an example of anassociation of guided (asymmetric) interaction pairs, meaning that themembers of the pair associate preferentially with each other rather thanself-associate. FIG. 10B is an example of an association of unguidedinteraction pairs, meaning that the members of the pair may associatewith each other or self-associate without substantial preference and mayhave the same or different amino acid sequences.

Such antibody-ALK7:ActRIIB complexes may be useful in situations whereit is desirable to further bind/antagonize an agent that is not anALK7:ActRIIB ligand. Alternatively, such antibody-ALK7:ActRIIB complexesmay be useful in situations where it is desirable to further enhanceALK7:ActRIIB ligand binding/antagonism. For example, as demonstrated bythe examples herein, an ALK7:ActRIIB heterodimer has high affinity forseveral ligands including, e.g., activin B and activin AC. In addition,ALK7:ActRIIB heterodimers bind to GDF3 but with weaker affinity. Incertain situations where it is desirable to antagonize GDF3 activity inaddition to one or more of the high affinity-binding ligands (e.g.,activin B and activin AC), GDF3 may be outcompeted for binding to anALK7:ActRIIB heterodimer. In these situations, addition of bindingdomain of an anti-GDF3 antibody to the ALK7:ActRIIB heteromultimercomplex would improve the capacity of the protein complex to antagonizeGDF3 in addition to one or more of the higher affinity ALK7:ActRIIBbinding-ligands (e.g., activin B, and activin AC).

FIG. 11 shows schematic examples of ALK7:ActRIIB single-trappolypeptides. ALK7:ActRIIB single-trap polypeptides may contain multipleALK7 domains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains),having the same or different sequences, and multiple ActRIIB domains(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains), having the sameor different sequences. These ALK7 and ActRIIB domains may be arrangedin any order and may comprise one or more linker domains positionsbetween one or more of the ALK7 and ActRIIB domains. Such ligand trapsmay be useful as therapeutic agents to treat or prevent diseases ordisorders described herein.

FIG. 12A-12D show schematic examples of multimeric protein complexescomprising at least one ALK7:ActRIIB single-chain trap polypeptide. Inthe illustrated embodiments 12A and 12B, a first ALK7:ActRIIBsingle-chain trap polypeptide (from left to right) is part of a fusionpolypeptide that comprises a first member of an interaction pair (“C₁”);and a second ALK7:ActRIIB single-chain trap polypeptide is part of afusion polypeptide that comprises a second member of an interaction pair(“C₂”). C₁ and C₂ may be the same or different. The first and secondALK7:ActRIIB single-chain trap polypeptides may be the same ordifferent. In each fusion polypeptide, a linker may be positionedbetween the ALK7:ActRIIB single-chain trap polypeptide and thecorresponding member of the interaction pair. Suitable interaction pairsincluded, for example, heavy chain and/or light chain immunoglobulininteraction pairs, truncations, and variants thereof as described herein[e.g., Spiess et al (2015) Molecular Immunology 67(2A): 95-106]. FIG.12A is an example of an association of unguided interaction pairs,meaning that the members of the pair may associate with each other orself-associate without substantial preference and may have the same ordifferent amino acid sequences. FIG. 12B is an example of an associationof guided (asymmetric) interaction pairs, meaning that the members ofthe pair associate preferentially with each other rather thanself-associate. Complexes of higher order can be envisioned. Inaddition, such ALK7:ActRIIB single-chain trap polypeptides may besimilarly be associated, covalently or non-covalently, with one or moreALK7 polypeptides and/or one or more ActRIIB polypeptides. See FIG. 12C.Also, such ALK7:ActRIIB single-chain trap polypeptides may be similarlybe associated, covalently or non-covalently, with one or moreligand-binding domain of an antibody (e.g., a ligand binding domain ofan antibody that binds to one or more ALK7:ActRIIB binding-ligands). SeeFIG. 12D.

FIGS. 13A-13C shows gene expression profiles of fibrotic genes (Col1a1,Col3a1, Fibronectin, PAI-1, CTGF, and a-SMA), inflammatory genes (Tnfa,and MCP1), cytokine genes (Tgfb1, Tgfb2, Tgfb3, and activin A), kidneyinjury gene (NGAL), Hypoxia-inducible factor 1-alpha (HIF1a), andactivin A receptor (Acvr2A) from mouse kidneys subjected to unilateralureteral obstruction (UUO). Samples from the contralateral, non-surgerykidney were used as a control (Ctrl). Gene expression profiles wereobtained at 3 days and 17 days post-surgery. Mice were administeredeither PBS or an ALK7-Fc:ActRIIB-Fc homodimer at days 3, 7, 10, and 14post-surgery. Statistics was performed using a one-way ANOVA followed byTukey analysis. (*) denotes a statistical difference between i) controlsamples compared to UUO kidneys at 3 days or ii) control samplescompared to UUO kidneys at 17 days in mice administered theALK7-Fc:ActRIIB-Fc homodimer. ($) denotes a statistical differencebetween UUO kidneys at 17 days in mice administered only PBS comparedwith UUO kidneys at 17 days in mice administered the ALK7-Fc:ActRIIB-Fchomodimer. (@) denotes that no transcript was detected.

DETAIL DESCRIPTION OF THE INVENTION

1. Overview

In part, the present disclosure relates to heteromultimers comprising aTGFβ superfamily type I receptor polypeptide and a TGFβ superfamily typeII receptor polypeptide, uses thereof, and methods of making suchheteromultimers. See, e.g., FIGS. 1 and 2 . In certain preferredembodiments, heteromultimers comprise an extracellular domain of a TGFβsuperfamily type I receptor polypeptide and an extracellular domain of aTGFβ superfamily type II receptor polypeptide. In particular, thedisclosure provides heteromultimers comprising an ALK7 polypeptide andan ActRIIB polypeptide. Preferably ALK7 polypeptides comprise aligand-binding domain of an ALK7 receptor, and ActRIIB polypeptidescomprise a ligand-binding domain of an ActRIIB receptor. In certainpreferred embodiments, ALK7:ActRIIB heteromultimers have an altered TGFβsuperfamily ligand binding profile/specificity compared to acorresponding sample of a homomultimer (e.g., an ALK7:ActRIIBheterodimer compared to an ActRIIB:ActRIIB homodimer or an ALK7:ALK7homodimer).

The TGF-β superfamily is comprised of over 30 secreted factors includingTGF-betas, activins, nodals, bone morphogenetic proteins (BMPs), growthand differentiation factors (GDFs), and anti-Mullerian hormone (AMH)[Weiss et al. (2013) Developmental Biology, 2(1): 47-63]. Members of thesuperfamily, which are found in both vertebrates and invertebrates, areubiquitously expressed in diverse tissues and function during theearliest stages of development throughout the lifetime of an animal.Indeed, TGF-β superfamily proteins are key mediators of stem cellself-renewal, gastrulation, differentiation, organ morphogenesis, andadult tissue homeostasis. Consistent with this ubiquitous activity,aberrant TGF-beta superfamily signaling is associated with a wide rangeof human pathologies including, for example, autoimmune disease,cardiovascular disease, fibrotic disease, and cancer.

Ligands of the TGF-beta superfamily share the same dimeric structure inwhich the central 3½ turn helix of one monomer packs against the concavesurface formed by the beta-strands of the other monomer. The majority ofTGF-beta family members are further stabilized by an intermoleculardisulfide bond. This disulfide bonds traverses through a ring formed bytwo other disulfide bonds generating what has been termed a ‘cysteineknot’ motif [Lin et al. (2006) Reproduction 132: 179-190; and Hinck etal. (2012) FEBS Letters 586: 1860-1870].

TGF-beta superfamily signaling is mediated by heteromeric complexes oftype I and type II serine/threonine kinase receptors, whichphosphorylate and activate downstream SMAD proteins (e.g., SMAD proteins1, 2, 3, 5, and 8) upon ligand stimulation [Massague (2000) Nat. Rev.Mol. Cell Biol. 1:169-178]. These type I and type II receptors aretransmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine kinase specificity.In general, type I receptors mediate intracellular signaling while thetype II receptors are required for binding TGF-beta superfamily ligands.Type I and II receptors form a stable complex after ligand binding,resulting in phosphorylation of type I receptors by type II receptors.

The TGF-beta family can be divided into two phylogenetic branches basedon the type I receptors they bind and the Smad proteins they activate.One is the more recently evolved branch, which includes, e.g., theTGF-betas, activins, GDF8, GDF9, GDF11, BMP3 and nodal, which signalthrough type I receptors that activate Smads 2 and 3 [Hinck (2012) FEBSLetters 586:1860-1870]. The other branch comprises the more distantlyrelated proteins of the superfamily and includes, e.g., BMP2, BMP4,BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF1, GDF5, GDF6, and GDF7,which signal through Smads 1, 5, and 8.

TGF-beta isoforms are the founding members of the TGF-beta superfamily,of which there are 3 known isoforms in mammals designated as TGF-beta1,TGF-beta2 and TGF-beta3. Mature bioactive TGF-beta ligands function ashomodimers and predominantly signal through the type I receptor ALK5,but have also been found to additionally signal through ALK1 inendothelial cells [Goumans et al. (2003) Mol Cell 12(4): 817-828].TGF-beta1 is the most abundant and ubiquitously expressed isoform.TGF-beta1 is known to have an important role in wound healing, and miceexpressing a constitutively active TGF-beta1 transgene develop fibrosis[Clouthier et al. (1997) J Clin. Invest. 100(11): 2697-2713]. TGF-beta1is also involved in T cell activation and maintenance of T regulatorycells [Li et al. (2006) Immunity 25(3): 455-471]. TGF-beta2 expressionwas first described in human glioblastoma cells, and is occurs inneurons and astroglial cells of the embryonic nervous system. TGF-beta2is known to suppress interleukin-2-dependent growth of T lymphocytes.TGF-beta3 was initially isolated from a human rhabdomyosarcoma cell lineand since has been found in lung adenocarcinoma and kidney carcinomacell lines. TGF-beta3 is known to be important for palate and lungmorphogenesis [Kubiczkova et al. (2012) Journal of TranslationalMedicine 10:183].

Activins are members of the TGF-beta superfamily and were initiallydiscovered as regulators of secretion of follicle-stimulating hormone,but subsequently various reproductive and non-reproductive roles havebeen characterized. There are three principal activin forms (A, B, andAB) that are homo/heterodimers of two closely related β subunits(β_(A)β_(A), β_(B)β_(B), and β_(A)β_(B), respectively). The human genomealso encodes an activin C and an activin E, which are primarilyexpressed in the liver, and heterodimeric forms containing Pc or PE arealso known. In the TGF-beta superfamily, activins are unique andmultifunctional factors that can stimulate hormone production in ovarianand placental cells, support neuronal cell survival, influencecell-cycle progress positively or negatively depending on cell type, andinduce mesodermal differentiation at least in amphibian embryos [DePaoloet al. (1991) Proc Soc Ep Biol Med. 198:500-512; Dyson et al. (1997)Curr Biol. 7:81-84; and Woodruff (1998) Biochem Pharmacol. 55:953-963].In several tissues, activin signaling is antagonized by its relatedheterodimer, inhibin. For example, in the regulation offollicle-stimulating hormone (FSH) secretion from the pituitary, activinpromotes FSH synthesis and secretion, while inhibin reduces FSHsynthesis and secretion. Other proteins that may regulate activinbioactivity and/or bind to activin include follistatin (FS),follistatin-related protein (FSRP, also known as FLRG or FSTL3), andα₂-macroglobulin.

As described herein, agents that bind to “activin A” are agents thatspecifically bind to the β_(A) subunit, whether in the context of anisolated β_(A) subunit or as a dimeric complex (e.g., a β_(A)β_(A)homodimer or a β_(A)β_(B) heterodimer). In the case of a heterodimercomplex (e.g., a β_(A)β_(B) heterodimer), agents that bind to “activinA” are specific for epitopes present within the β_(A) subunit, but donot bind to epitopes present within the non-β_(A) subunit of the complex(e.g., the β_(B) subunit of the complex). Similarly, agents disclosedherein that antagonize (inhibit) “activin A” are agents that inhibit oneor more activities as mediated by a β_(A) subunit, whether in thecontext of an isolated β_(A) subunit or as a dimeric complex (e.g., aβ_(A)β_(A) homodimer or a β_(A)β_(B) heterodimer). In the case ofβ_(A)β_(B) heterodimers, agents that inhibit “activin A” are agents thatspecifically inhibit one or more activities of the β_(A) subunit, but donot inhibit the activity of the non-β_(A) subunit of the complex (e.g.,the β_(B) subunit of the complex). This principle applies also to agentsthat bind to and/or inhibit “activin B”, “activin C”, and “activin E”.Agents disclosed herein that antagonize “activin AB” are agents thatinhibit one or more activities as mediated by the β_(A) subunit and oneor more activities as mediated by the β_(B) subunit. The same principlealso applies to agents that bind to and/or inhibit “activin AC”,“activin AE”, “activin BC” or “activin BE”.

Nodal proteins have functions in mesoderm and endoderm induction andformation, as well as subsequent organization of axial structures suchas heart and stomach in early embryogenesis. It has been demonstratedthat dorsal tissue in a developing vertebrate embryo contributespredominantly to the axial structures of the notochord and pre-chordalplate while it recruits surrounding cells to form non-axial embryonicstructures. Nodal appears to signal through both type I and type IIreceptors and intracellular effectors known as SMAD proteins. Studiessupport the idea that ActRIIA and ActRIIB serve as type II receptors fornodal [Sakuma et al. (2002) Genes Cells. 2002, 7:401-12]. It issuggested that Nodal ligands interact with their co-factors (e.g.,Crypto or Cryptic) to activate activin type I and type II receptors,which phosphorylate SMAD2. Nodal proteins are implicated in many eventscritical to the early vertebrate embryo, including mesoderm formation,anterior patterning, and left-right axis specification. Experimentalevidence has demonstrated that nodal signaling activates pAR3-Lux, aluciferase reporter previously shown to respond specifically to activinand TGF-beta. However, nodal is unable to induce pTlx2-Lux, a reporterspecifically responsive to bone morphogenetic proteins. Recent resultsprovide direct biochemical evidence that nodal signaling is mediated bySMAD2 and SMAD3, which also mediate signaling by TGF-betas and activins.Further evidence has shown that the extracellular protein Cripto orCryptic is required for nodal signaling, making it distinct from activinor TGF-beta signaling.

The BMPs and GDFs together form a family of cysteine-knot cytokinessharing the characteristic fold of the TGF-beta superfamily [Rider etal. (2010) Biochem J., 429(1):1-12]. This family includes, for example,BMP2, BMP4, BMP6, BMP7, BMP2a, BMP3, BMP3b (also known as GDF10), BMP4,BMP5, BMP6, BMP7, BMP8, BMP8a, BMP8b, BMP9 (also known as GDF2), BMP10,BMP11 (also known as GDF11), BMP12 (also known as GDF7), BMP13 (alsoknown as GDF6), BMP14 (also known as GDF5), BMP15, GDF1, GDF3 (alsoknown as VGR2), GDF8 (also known as myostatin), GDF9, GDF15, anddecapentaplegic. Besides the ability to induce bone formation, whichgave the BMPs their name, the BMP/GDFs display morphogenetic activitiesin the development of a wide range of tissues. BMP/GDF homo- andhetero-dimers interact with combinations of type I and type II receptordimers to produce multiple possible signaling complexes, leading to theactivation of one of two competing sets of SMAD transcription factors.BMP/GDFs have highly specific and localized functions. These areregulated in a number of ways, including the developmental restrictionof BMP/GDF expression and through the secretion of several specific BMPantagonist proteins that bind with high affinity to the cytokines.Curiously, a number of these antagonists resemble TGF-beta superfamilyligands.

Growth and differentiation factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass and is highlyexpressed in developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of skeletal muscle [McPherron et al. Nature (1997)387:83-90]. Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle and, strikingly, inhumans [Ashmore et al. (1974) Growth, 38:501-507; Swatland and Kieffer,J. Anim. Sci. (1994) 38:752-757; McPherron and Lee, Proc. Natl. Acad.Sci. USA (1997) 94:12457-12461; Kambadur et al. Genome Res. (1997)7:910-915; and Schuelke et al. (2004) N Engl J Med, 350:2682-8]. Studieshave also shown that muscle wasting associated with HIV-infection inhumans is accompanied by increases in GDF8 protein expression[Gonzalez-Cadavid et al., PNAS (1998) 95:14938-43]. In addition, GDF8can modulate the production of muscle-specific enzymes (e.g., creatinekinase) and modulate myoblast cell proliferation [International PatentApplication Publication No. WO 00/43781]. The GDF8 propeptide cannoncovalently bind to the mature GDF8 domain dimer, inactivating itsbiological activity [Miyazono et al. (1988) J. Biol. Chem., 263:6407-6415; Wakefield et al. (1988) J. Biol. Chem., 263; 7646-7654; andBrown et al. (1990) Growth Factors, 3: 35-43]. Other proteins which bindto GDF8 or structurally related proteins and inhibit their biologicalactivity include follistatin, and potentially, follistatin-relatedproteins [Gamer et al. (1999) Dev. Biol., 208: 222-232].

GDF11, also known as BMP11, is a secreted protein that is expressed inthe tail bud, limb bud, maxillary and mandibular arches, and dorsal rootganglia during mouse development [McPherron et al. (1999) Nat. Genet.,22: 260-264; and Nakashima et al. (1999) Mech. Dev., 80: 185-189]. GDF11plays a unique role in patterning both mesodermal and neural tissues[Gamer et al. (1999) Dev Biol., 208:222-32]. GDF11 was shown to be anegative regulator of chondrogenesis and myogenesis in developing chicklimb [Gamer et al. (2001) Dev Biol., 229:407-20]. The expression ofGDF11 in muscle also suggests its role in regulating muscle growth in asimilar way to GDF8. In addition, the expression of GDF11 in brainsuggests that GDF11 may also possess activities that relate to thefunction of the nervous system. Interestingly, GDF11 was found toinhibit neurogenesis in the olfactory epithelium [Wu et al. (2003)Neuron., 37:197-207]. Hence, inhibitors of GDF11 may have in vitro andin vivo applications in the treatment of diseases such as musclediseases and neurodegenerative diseases (e.g., amyotrophic lateralsclerosis).

BMP7, also called osteogenic protein-1 (OP-1), is well known to inducecartilage and bone formation. In addition, BMP7 regulates a wide arrayof physiological processes. For example, BMP7 may be the osteoinductivefactor responsible for the phenomenon of epithelial osteogenesis. It isalso found that BMP7 plays a role in calcium regulation and bonehomeostasis. Like activin, BMP7 binds to type II receptors, ActRIIA andActRIIB However, BMP7 and activin recruit distinct type I receptors intoheteromeric receptor complexes. The major BMP7 type I receptor observedwas ALK2, while activin bound exclusively to ALK4 (ActRIIB) BMP7 andactivin elicited distinct biological responses and activated differentSMAD pathways [Macias-Silva et al. (1998) J Biol Chem. 273:25628-36].

As described herein, comparative binding data demonstrated that anALK7:ActRIIB heterodimer has an altered binding profile (ligandselectivity) compared to either corresponding ActRIIB or ALK7homodimers. In particular, the ALK7:ActRIIB heterodimer displaysenhanced binding to activin C, activin AC, and BMP5 compared to eitherhomodimer, and retains strong binding to activin B as observed with theActRIIB homodimer. However, the ALK7:ActRIIB heterodimer exhibitsreduced binding to GDF11, GDF8, activin A, BMP10, BMP6, GDF3, and BMP9compared to the ActRIIB homodimer. In particular, BMP9 displays low orno observable affinity for the ALK7:ActRIIB heterodimer, whereas thisligand binds tightly to ActRIIB homodimer.

These results therefore demonstrate that ALK7:ActRIIB heterodimers aremore selective antagonists of activin B, activin C, activin AC, and BMP5compared to ActRIIB homodimers. Additionally, it is expected that suchheterodimers will bind tightly to Activin BC, given the binding toActivins B and C. Additionally, it is expected that such heterodimerswill bind to activin E, activin AE and activin BE given the structuralsimilarity between activin C and E. Accordingly, an ALK7:ActRIIBheterodimer may be more useful than an ActRIIB homodimer in certainapplications where such selective antagonism is advantageous. Examplesinclude therapeutic applications where it is desirable to retainantagonism of one or more of activin B, activin C, activin AC, activinBC, activin E, activin AE, activin BE and BMP5 but minimize antagonismof one or more of activin A, BMP9, BMP10, GDF3, and BMP6.

Moreover, ALK7:ActRIIB heterodimers, as described herein, exertbeneficial catabolic effects on adipose tissue. Additionally, theALK7:ActRIIB heterodimers described herein are shown to have potentprotective effects in a model of chronic kidney disease, with effectsincluding the inhibition of fibrosis and inflammation. However, unlikeActRIIB homodimer, an ActRIIB:ALK7 heterodimer exhibits onlylow-affinity or transient binding to BMP9 and so will have little to noconcurrent inhibition on processes mediated by BMP9, such asangiogenesis. This novel selectivity will be useful, for example, intreating patients in need of inhibitory effects on fat, or protectiveeffects on the kidney, but not in need of altered angiogenesis.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this disclosure and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification to provide additional guidanceto the practitioner in describing the compositions and methods of thedisclosure and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which it isused.

The terms “heteromer” or “heteromultimer” is a complex comprising atleast a first polypeptide chain and a second polypeptide chain, whereinthe second polypeptide chain differs in amino acid sequence from thefirst polypeptide chain by at least one amino acid residue. Theheteromer can comprise a “heterodimer” formed by the first and secondpolypeptide chains or can form higher order structures where one or morepolypeptide chains in addition to the first and second polypeptidechains are present. Exemplary structures for the heteromultimer includeheterodimers, heterotrimers, heterotetramers and further oligomericstructures. Heterodimers are designated herein as X:Y or equivalently asX-Y, where X represents a first polypeptide chain and Y represents asecond polypeptide chain. Higher-order heteromers and oligomericstructures are designated herein in a corresponding manner. In certainembodiments a heteromultimer is recombinant (e.g., one or morepolypeptide components may be a recombinant protein), isolated and/orpurified.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions. However, in common usage andin the instant application, the term “homologous,” when modified with anadverb such as “highly,” may refer to sequence similarity and may or maynot relate to a common evolutionary origin.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

“Percent (%) sequence identity” with respect to a reference polypeptide(or nucleotide) sequence is defined as the percentage of amino acidresidues (or nucleic acids) in a candidate sequence that are identicalto the amino acid residues (or nucleic acids) in the referencepolypeptide (nucleotide) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid (nucleic acid) sequenceidentity values are generated using the sequence comparison computerprogram ALIGN-2. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc., and the source code has been filed withuser documentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available from Genentech, Inc., SouthSan Francisco, Calif., or may be compiled from the source code. TheALIGN-2 program should be compiled for use on a UNIX operating system,including digital UNIX V4.0D. All sequence comparison parameters are setby the ALIGN-2 program and do not vary.

“Agonize”, in all its grammatical forms, refers to the process ofactivating a protein and/or gene (e.g., by activating or amplifying thatprotein's gene expression or by inducing an inactive protein to enter anactive state) or increasing a protein's and/or gene's activity.

“Antagonize”, in all its grammatical forms, refers to the process ofinhibiting a protein and/or gene (e.g., by inhibiting or decreasing thatprotein's gene expression or by inducing an active protein to enter aninactive state) or decreasing a protein's and/or gene's activity.

As used herein, unless otherwise stated, “does not substantially bind toX” is intended to mean that an agent has a K_(D) that is greater thanabout 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴, or greater (e.g., no detectable binding bythe assay used to determine the K_(D)) for “X”, wherein “X” is aspecified agent such as protein or nucleic acid.

The terms “about” and “approximately” as used in connection with anumerical value throughout the specification and the claims denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart. In general, such interval of accuracy is ±10%. Alternatively, andparticularly in biological systems, the terms “about” and“approximately” may mean values that are within an order of magnitude,preferably ≤5-fold and more preferably ≤2-fold of a given value.

Numeric ranges disclosed herein are inclusive of the numbers definingthe ranges.

The terms “a” and “an” include plural referents unless the context inwhich the term is used clearly dictates otherwise. The terms “a” (or“an”), as well as the terms “one or more,” and “at least one” can beused interchangeably herein. Furthermore, “and/or” where used herein isto be taken as specific disclosure of each of the two or more specifiedfeatures or components with or without the other. Thus, the term“and/or” as used in a phrase such as “A and/or B” herein is intended toinclude “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, theterm “and/or” as used in a phrase such as “A, B, and/or C” is intendedto encompass each of the following aspects: A, B, and C; A, B, or C; Aor C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone);and C (alone).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

2. ActRIIB:ALK7 Antagonists

As described herein, it has been discovered that ALK7:ActRIIBheterodimers have a unique ligand-binding profile/selectivity comparedto corresponding ActRIIB and ALK7 homodimers. Interestingly, four of thefive ligands with the strongest binding to ActRIIB homodimer (activin A,BMP10, GDF8, and GDF11) exhibit reduced binding to the ALK7:ActRIIBheterodimer, the exception being activin B which retains tight bindingto the heterodimer. Similarly, three of the four ligands withintermediate binding to ActRIIB homodimer (GDF3, BMP6, and particularlyBMP9) exhibit reduced binding to the ALK7:ActRIIB heterodimer, whereasbinding to activin AC is increased to become the second strongest ligandinteraction with the heterodimer overall. Finally, activin C and BMP5unexpectedly bind the ALK7:ActRIIB heterodimer with intermediatestrength despite no binding (activin C) or weak binding (BMP5) toActRIIB homodimer. Given the binding to activin B and activin C, it isexpected that the ALK7:ActRIIB heterodimer binds tightly to activin BC.The net result is that the ALK7:ActRIIB heterodimer possesses aligand-binding profile distinctly different from that of either ActRIIBhomodimer or ALK7 homodimer, which binds none of the foregoing ligands.

These results therefore indicate that the ALK7:ActRIIB heteromultimersare more selective antagonist of activin B, activin AC and Activin BCcompared to ActRIIB homomultimers. Moreover, ALK7:ActRIIB heterodimerexhibits the unusual property of robust binding to activin C.Accordingly, an ALK7:ActRIIB heteromultimers will be more useful than anActRIIB homomultimers in certain applications where such selectiveantagonism is advantageous.

Moreover, ALK7:ActRIIB heteromultimers are surprisingly effective inameliorating various complications of kidney disease (e.g., treating orpreventing kidney injury, inflammation, and damage) as well as exertingbeneficial anabolic adipose cells. Although ALK7:ActRIIB heteromultimersmay exert biological effects through a mechanism other than ligandinhibition [e.g., inhibition of one or more of activin B, activin AC,activin C, GDF11, GDF8, activin A, BMP10, BMP6, BMP5, nodal, and GDF3may be an indicator of the tendency of an agent to inhibit theactivities of a spectrum of additional agents, including, perhaps, othermembers of the TGF-beta superfamily, and such collective inhibition maylead to a desired effect on, for example, kidney disease or a metabolicdisorder], other types of TGF-beta superfamily ligand antagonists aswell as type I and type II receptor antagonists (e.g., natural ligandtraps such as follistatin and Lefty, antibodies, inhibitory nucleicacids, and inhibitory small molecules), or combinations of suchantagonists, are expected to be useful in accordance with the methodsdescribed herein, particularly those that that mimic thebinding/inhibitory properties of the ALK7:ActRIIB heterodimers as wellas agents that directly or indirectly antagonize ALK7 and/or ActRIIBreceptors, agents that directly or indirectly antagonize ALK7 and/orActRIIB-binding ligands, agents that directly or indirectly antagonizedownstream signaling mediators (e.g., Smads), and/or agents thatdirectly or indirectly antagonize TGFβ superfamily co-receptors (e.g.,Cripto or Cryptic) will have similar biological effects. CollectivelyALK7:ActRIIB heteromultimers and these alternative antagonists arereferred to herein as “ALK7:ActRIIB antagonists” or “ALK7:ActRIIBinhibitors”.

A. ALK7:ActRIIB Heteromultimers

In certain aspects, the present disclosure relates to heteromultimerscomprising one or more ALK7 receptor polypeptides (e.g., SEQ ID NOs: 9,10, 19, 20, 38, 39, 42, 43, 46, 74, 76, 79, and 80) and one or moreActRIIB receptor polypeptides (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 71,73, 77, and 78), which are generally referred to herein as “ALK7:ActRIIBheteromultimer complexes” or “ALK7:ActRIIB heteromultimers”. Preferably,ALK7:ActRIIB heteromultimers of the disclosure are soluble, for example,a heteromultimer may comprises a soluble portion (domain) of an ALK7receptor and a soluble portion (domain) of an ActRIIB receptor. Ingeneral, the extracellular domains of ALK7 and ActRIIB correspond to asoluble portion of these receptors. Therefore, in some embodiments,heteromultimers of the disclosure comprise an extracellular domain of anALK7 receptor and an extracellular domain of an ActRIIB receptor.Example extracellular domains of ALK7 and ActRIIB receptors aredisclosed herein and these sequences, as well as fragments, functionalvariants, and modified forms thereof, may be used in accordance with theinventions of the disclosure (e.g., ALK7:ActRIIB heteromultimers anduses thereof). ALK7:ActRIIB heteromultimers of the disclosure include,e.g., heterodimers, heterotrimers, heterotetramers and higher orderoligomeric structures. See, e.g., FIGS. 1, 2, and 8-10 . In certainpreferred embodiments, heteromultimers of the disclosure areALK7:ActRIIB heterodimers.

Preferably, ALK7:ActRIIB heteromultimers of the disclosure bind to oneor more TGF-beta superfamily ligands. In some embodiments, ALK7:ActRIIBheteromultimers bind to one or more of activin (e.g., activin A, activinB, activin C, activin E, activin AB, activin AC, activin AE, activin BC,and activin BE), GDF8, and GDF11. Optionally, in some embodiments,ALK7:ActRIIB heteromultimers further bind to BMP6. Optionally, in someembodiments, ALK7:ActRIIB heteromultimers further bind to BMP10.Optionally, in some embodiments, ALK7:ActRIIB heteromultimers furtherbind to BMP5. Optionally, in some embodiments, ALK7:ActRIIBheteromultimers further bind to GDF3. Optionally, in some embodiments,ALK7:ActRIIB heteromultimers further bind to nodal. Optionally, in someembodiments, ALK7:ActRIIB heteromultimers further bind to BMP10.Optionally, in some embodiments, ALK7:ActRIIB heteromultimers do notbind or do not substantially bind to BMP9. In certain preferredembodiments, ALK7:ActRIIB heteromultimers bind one or more of GDF11,GDF8, activin A, BMP10, BMP6, GDF3, and BMP9 with weaker affinitycompared to a corresponding ActRIIB homomultimer. In other preferredembodiments, ALK7:ActRIIB heteromultimers bind one or more of activin C,activin AC, activin BC, and BMP5 with stronger affinity compared to acorresponding ActRIIB homomultimer. Optionally, ALK7:ActRIIBheteromultimers further bind to one or more of BMP2, BMP2/7, BMP3, BMP4,BMP4/7, BMP7, BMP8a, BMP8b, GDF5, GDF6/BMP13, GDF7, GDF9b/BMP15,GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, glial cell-derived neurotrophicfactor (GDNF), neurturin, artemin, persephin, MIS, and Lefty.

TGF-beta superfamily ligand-binding ALK7:ActRIIB heteromultimers may beused to inhibit (antagonize) signaling (e.g., Smad 2/3 and/or Smad 1/5/8signaling) mediated by one or more TGFβ superfamily ligands. Inparticular, ALK7:ActRIIB heteromultimers of the disclosure may be usedto inhibit signaling by one or more TGFβ superfamily ligands in, forexample, a cell-based assay such as those described herein. For example,ALK7:ActRIIB heteromultimers inhibit signaling mediated by one or moreof e.g., activin (e.g., activin A, activin B, activin C, activin E,activin AB, activin AC, activin BC, activin AE and activin BE), GDF11,GDF8, BMP10, BMP6, BMP5, nodal, and GDF3 in a cell-based assay.Optionally, in some embodiments, ALK7:ActRIIB heteromultimers mayfurther inhibit BMP6 signaling in a cell-based assay. Optionally, insome embodiments, ALK7:ActRIIB heteromultimers may further inhibit GDF3signaling in a cell-based assay. Optionally, in some embodiments,ALK7:ActRIIB heteromultimers may further inhibit nodal signaling in acell-based assay. Optionally, in some embodiments, ALK7:ActRIIBheteromultimers may further inhibit BMP5 signaling in a cell-basedassay. Optionally, in some embodiments, ALK7:ActRIIB heteromultimers mayfurther inhibit signaling BMP10 signaling in a cell-based assay.Optionally, in some embodiments, ALK7:ActRIIB heteromultimers do notinhibit or do not substantially inhibit signaling mediated by BMP9 in acell-based assay. In certain preferred embodiments, ALK7:ActRIIBheteromultimers have a weaker inhibitory effect on signaling mediated byone or more of GDF11, GDF8, activin A, BMP10, BMP6, GDF3, and BMP9compared to a corresponding ActRIIB homomultimer in a cell-based assay.In other preferred embodiments, ALK7:ActRIIB heteromultimers have astronger inhibitory effect on signaling mediated by one or more ofactivin AC, activin BC, activin C, and BMP5 compared to a correspondingActRIIB homomultimer in a cell-based assay. Optionally, ALK7:ActRIIBheteromultimers may further inhibit signaling by one or more of BMP2,BMP2/7, BMP3, BMP4, BMP4/7, BMP7, BMP8a, BMP8b, GDF5, GDF6/BMP13, GDF7,GDF9b/BMP15, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, glial cell-derivedneurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, andLefty in a cell-based assay.

As used herein, the term “ActRIIB” refers to a family of activinreceptor type IIB (ActRIIB) proteins from any species and variantsderived from such ActRIIB proteins by mutagenesis or other modification.Reference to ActRIIB herein is understood to be a reference to any oneof the currently identified forms. Members of the ActRIIB family aregenerally transmembrane proteins, composed of a ligand-bindingextracellular domain comprising a cysteine-rich region, a transmembranedomain, and a cytoplasmic domain with predicted serine/threonine kinaseactivity.

The term “ActRIIB polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIB family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Examples of suchvariant ActRIIB polypeptides are provided throughout the presentdisclosure as well as in International Patent Application PublicationNos. WO 2006/012627, WO 2008/097541, and WO 2010/151426, which areincorporated herein by reference in their entirety. Numbering of aminoacids for all ActRIIB-related polypeptides described herein is based onthe numbering of the human ActRIIB precursor protein sequence providedbelow (SEQ ID NO: 1), unless specifically designated otherwise.

The human ActRIIB precursor protein sequence is as follows:

(SEQ ID NO: 1)   1 MTAPWVALAL LWGSLCAGS G RGEAETRECI YYNANWELER T

QSGLERCE  51 GEQDKRLHCY ASWR

SSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY 101FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS 151LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR 201FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA 251EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY 301LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK 351PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC 401KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL 451AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV 501 TNVDLPPKES SI

The signal peptide is indicated with a single underline; theextracellular domain is indicated in bold font; and the potential,endogenous N-linked glycosylation sites are indicated with a doubleunderline.

The processed (mature) extracellular ActRIIBpolypeptide sequence is as follows: (SEQ ID NO: 2)GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLP EAGGPEVTYEPPPTAPT

In some embodiments, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The C-terminal “tail” of the extracellulardomain is indicated by a single underline. The sequence with the “tail”deleted (a Δ15 sequence) is as follows:

(SEQ ID NO: 3) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA

A form of ActRIIB with an alanine at position 64 of SEQ ID NO: 1 (A64)is also reported in the literature. See, e.g., Hilden et al. (1994)Blood, 83(8): 2163-2170. Applicants have ascertained that an ActRIIB-Fcfusion protein comprising an extracellular domain of ActRIIB with theA64 substitution has a relatively low affinity for activin and GDF11. Bycontrast, the same ActRIIB-Fc fusion protein with an arginine atposition 64 (R64) has an affinity for activin and GDF11 in the lownanomolar to high picomolar range. Therefore, sequences with an R64 areused as the “wild-type” reference sequence for human ActRIIB in thisdisclosure.

The form of ActRIIB with an alanine at position 64 is as follows:

(SEQ ID NO: 4) 1 MTAPWVALAL LWGSLCAGS G RGEAETRECI YYNANWELERTNQSGLERCE  51 GEQDKRLHCY ASWANSSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY 101 FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS  151LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR  201FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA  251EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY  301LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK  351PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC  401KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL  451AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV  501TNVDLPPKES SI 

The signal peptide is indicated by single underline and theextracellular domain is indicated by bold font.

The processed (mature) extracellular ActRIIB polypeptide sequence of thealternative A64 form is as follows:

(SEQ ID NO: 5) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA GGPEVTYEPPPTAPT

In some embodiments, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The C-terminal “tail” of the extracellulardomain is indicated by single underline. The sequence with the “tail”deleted (a Δ15 sequence) is as follows:

(SEQ ID NO: 6) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA

A nucleic acid sequence encoding the human ActRIIB precursor protein isshown below (SEQ ID NO: 7), representing nucleotides 25-1560 of GenbankReference Sequence NM_001106.3, which encode amino acids 1-513 of theActRIIB precursor. The sequence as shown provides an arginine atposition 64 and may be modified to provide an alanine instead. Thesignal sequence is underlined.

(SEQ ID NO: 7) 1 ATGACGGCGC CCTGGGTGGC CCTCGCCCTC CTCTGGGGAT CGCTGTGCGC51 CGGCTCTGGG CGTGGGGAGG CTGAGACACG GGAGTGCATC TACTACAACG 101CCAACTGGGA GCTGGAGCGC ACCAACCAGA GCGGCCTGGA GCGCTGCGAA  151GGCGAGCAGG ACAAGCGGCT GCACTGCTAC GCCTCCTGGC GCAACAGCTC  201TGGCACCATC GAGCTCGTGA AGAAGGGCTG CTGGCTAGAT GACTTCAACT  251GCTACGATAG GCAGGAGTGT GTGGCCACTG AGGAGAACCC CCAGGTGTAC  301TTCTGCTGCT GTGAAGGCAA CTTCTGCAAC GAACGCTTCA CTCATTTGCC  351AGAGGCTGGG GGCCCGGAAG TCACGTACGA GCCACCCCCG ACAGCCCCCA  401CCCTGCTCAC GGTGCTGGCC TACTCACTGC TGCCCATCGG GGGCCTTTCC  451CTCATCGTCC TGCTGGCCTT TTGGATGTAC CGGCATCGCA AGCCCCCCTA  501CGGTCATGTG GACATCCATG AGGACCCTGG GCCTCCACCA CCATCCCCTC  551TGGTGGGCCT GAAGCCACTG CAGCTGCTGG AGATCAAGGC TCGGGGGCGC  601TTTGGCTGTG TCTGGAAGGC CCAGCTCATG AATGACTTTG TAGCTGTCAA  651GATCTTCCCA CTCCAGGACA AGCAGTCGTG GCAGAGTGAA CGGGAGATCT  701TCAGCACACC TGGCATGAAG CACGAGAACC TGCTACAGTT CATTGCTGCC  751GAGAAGCGAG GCTCCAACCT CGAAGTAGAG CTGTGGCTCA TCACGGCCTT  801CCATGACAAG GGCTCCCTCA CGGATTACCT CAAGGGGAAC ATCATCACAT  851GGAACGAACT GTGTCATGTA GCAGAGACGA TGTCACGAGG CCTCTCATAC  901CTGCATGAGG ATGTGCCCTG GTGCCGTGGC GAGGGCCACA AGCCGTCTAT  951TGCCCACAGG GACTTTAAAA GTAAGAATGT ATTGCTGAAG AGCGACCTCA  1001CAGCCGTGCT GGCTGACTTT GGCTTGGCTG TTCGATTTGA GCCAGGGAAA  1051CCTCCAGGGG ACACCCACGG ACAGGTAGGC ACGAGACGGT ACATGGCTCC  1101TGAGGTGCTC GAGGGAGCCA TCAACTTCCA GAGAGATGCC TTCCTGCGCA  1151TTGACATGTA TGCCATGGGG TTGGTGCTGT GGGAGCTTGT GTCTCGCTGC  1201AAGGCTGCAG ACGGACCCGT GGATGAGTAC ATGCTGCCCT TTGAGGAAGA  1251GATTGGCCAG CACCCTTCGT TGGAGGAGCT GCAGGAGGTG GTGGTGCACA  1301AGAAGATGAG GCCCACCATT AAAGATCACT GGTTGAAACA CCCGGGCCTG  1351GCCCAGCTTT GTGTGACCAT CGAGGAGTGC TGGGACCATG ATGCAGAGGC  1401TCGCTTGTCC GCGGGCTGTG TGGAGGAGCG GGTGTCCCTG ATTCGGAGGT  1451CGGTCAACGG CACTACCTCG GACTGTCTCG TTTCCCTGGT GACCTCTGTC  1501ACCAATGTGG ACCTGCCCCC TAAAGAGTCA AGCATC

A nucleic acid sequence encoding processed extracellular human ActRIIBpolypeptide is shown below (SEQ ID NO: 8). The sequence as shownprovides an arginine at position 64, and may be modified to provide analanine instead.

(SEQ ID NO: 8) 1 GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG 51 GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC  101AGGACAAGCG GCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC  151ATCGAGCTCG TGAAGAAGGG CTGCTGGCTA GATGACTTCA ACTGCTACGA  201TAGGCAGGAG TGTGTGGCCA CTGAGGAGAA CCCCCAGGTG TACTTCTGCT  251GCTGTGAAGG CAACTTCTGC AACGAACGCT TCACTCATTT GCCAGAGGCT  301GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACC 

An alignment of the amino acid sequences of human ActRIIB extracellulardomain and human ActRIIA extracellular domain are illustrated in FIG. 3. This alignment indicates amino acid residues within both receptorsthat are believed to directly contact ActRII ligands. For example, thecomposite ActRII structures indicated that the ActRIIB-ligand bindingpocket is defined, in part, by residues Y31, N33, N35, L38 through T41,E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83,Y85, R87, A92, and E94 through F101. At these positions, it is expectedthat conservative mutations will be tolerated.

In addition, ActRIIB is well-conserved among vertebrates, with largestretches of the extracellular domain completely conserved. For example,FIG. 4 depicts a multi-sequence alignment of a human ActRIIBextracellular domain compared to various ActRIIB orthologs. Many of theligands that bind to ActRIIB are also highly conserved. Accordingly,from these alignments, it is possible to predict key amino acidpositions within the ligand-binding domain that are important for normalActRIIB-ligand binding activities as well as to predict amino acidpositions that are likely to be tolerant of substitution withoutsignificantly altering normal ActRIIB-ligand binding activities.Therefore, an active, human ActRIIB variant polypeptide useful inaccordance with the presently disclosed methods may include one or moreamino acids at corresponding positions from the sequence of anothervertebrate ActRIIB, or may include a residue that is similar to that inthe human or other vertebrate sequences. Without meaning to be limiting,the following examples illustrate this approach to defining an activeActRIIB variant. L46 in the human extracellular domain (SEQ ID NO: 53)is a valine in Xenopus ActRIIB (SEQ ID NO: 55), and so this position maybe altered, and optionally may be altered to another hydrophobicresidue, such as V, I or F, or a non-polar residue such as A. E52 in thehuman extracellular domain is a K in Xenopus, indicating that this sitemay be tolerant of a wide variety of changes, including polar residues,such as E, D, K, R, H, S, T, P, G, Y and probably A. T93 in the humanextracellular domain is a K in Xenopus, indicating that a widestructural variation is tolerated at this position, with polar residuesfavored, such as S, K, R, E, D, H, G, P, G and Y. F108 in the humanextracellular domain is a Y in Xenopus, and therefore Y or otherhydrophobic group, such as I, V or L should be tolerated. E111 in thehuman extracellular domain is K in Xenopus, indicating that chargedresidues will be tolerated at this position, including D, R, K and H, aswell as Q and N. R112 in the human extracellular domain is K in Xenopus,indicating that basic residues are tolerated at this position, includingR and H. A at position 119 in the human extracellular domain isrelatively poorly conserved, and appears as P in rodents and V inXenopus, thus essentially any amino acid should be tolerated at thisposition.

Moreover, ActRII proteins have been characterized in the art in terms ofstructural and functional characteristics, particularly with respect toligand binding [Attisano et al. (1992) Cell 68(1):97-108; Greenwald etal. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al.(2006) PNAS 103(20: 7643-7648; Thompson et al. (2003) The EMBO Journal22(7): 1555-1566; as well as U.S. Pat. Nos. 7,709,605, 7,612,041, and7,842,663]. In addition to the teachings herein, these referencesprovide amply guidance for how to generate ActRIIB variants that retainone or more normal activities (e.g., ligand-binding activity).

For example, a defining structural motif known as a three-finger toxinfold is important for ligand binding by type I and type II receptors andis formed by conserved cysteine residues located at varying positionswithin the extracellular domain of each monomeric receptor [Greenwald etal. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett586:1860-1870]. Accordingly, the core ligand-binding domains of humanActRIIB, as demarcated by the outermost of these conserved cysteines,corresponds to positions 29-109 of SEQ ID NO: 1 (ActRIIB precursor).Thus, the structurally less-ordered amino acids flanking thesecysteine-demarcated core sequences can be truncated by about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, or 28 residues at the N-terminus and/or by about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 residues a the C-terminus without necessarily alteringligand binding. Exemplary ActRIIB extracellular domains for N-terminaland/or C-terminal truncation include SEQ ID NOs: 2, 3, 5, and 6.

Attisano et al. showed that a deletion of the proline knot at theC-terminus of the extracellular domain of ActRIIB reduced the affinityof the receptor for activin. An ActRIIB-Fc fusion protein containingamino acids 20-119 of present SEQ ID NO: 1, “ActRIIB(20-119)-Fc”, hasreduced binding to GDF11 and activin relative to an ActRIIB(20-134)-Fc,which includes the proline knot region and the complete juxtamembranedomain (see, e.g., U.S. Pat. No. 7,842,663). However, anActRIIB(20-129)-Fc protein retains similar, but somewhat reducedactivity, relative to the wild-type, even though the proline knot regionis disrupted.

Thus, ActRIIB extracellular domains that stop at amino acid 134, 133,132, 131, 130 and 129 (with respect to SEQ ID NO: 1) are all expected tobe active, but constructs stopping at 134 or 133 may be most active.Similarly, mutations at any of residues 129-134 (with respect to SEQ IDNO: 1) are not expected to alter ligand-binding affinity by largemargins. In support of this, it is known in the art that mutations ofP129 and P130 (with respect to SEQ ID NO: 1) do not substantiallydecrease ligand binding. Therefore, an ActRIIB polypeptide of thepresent disclosure may end as early as amino acid 109 (the finalcysteine), however, forms ending at or between 109 and 119 (e.g., 109,110, 111, 112, 113, 114, 115, 116, 117, 118, or 119) are expected tohave reduced ligand binding. Amino acid 119 (with respect to present SEQID NO: 1) is poorly conserved and so is readily altered or truncated.ActRIIB polypeptides ending at 128 (with respect to SEQ ID NO: 1) orlater should retain ligand-binding activity. ActRIIB polypeptides endingat or between 119 and 127 (e.g., 119, 120, 121, 122, 123, 124, 125, 126,or 127), with respect to SEQ ID NO: 1, will have an intermediate bindingability. Any of these forms may be desirable to use, depending on theclinical or experimental setting.

At the N-terminus of ActRIIB, it is expected that a protein beginning atamino acid 29 or before (with respect to SEQ ID NO: 1) will retainligand-binding activity. Amino acid 29 represents the initial cysteine.An alanine-to-asparagine mutation at position 24 (with respect to SEQ IDNO: 1) introduces an N-linked glycosylation sequence withoutsubstantially affecting ligand binding [U.S. Pat. No. 7,842,663]. Thisconfirms that mutations in the region between the signal cleavagepeptide and the cysteine cross-linked region, corresponding to aminoacids 20-29, are well tolerated. In particular, ActRIIB polypeptidesbeginning at position 20, 21, 22, 23, and 24 (with respect to SEQ IDNO: 1) should retain general ligand-biding activity, and ActRIIBpolypeptides beginning at positions 25, 26, 27, 28, and 29 (with respectto SEQ ID NO: 1) are also expected to retain ligand-biding activity. Ithas been demonstrated, e.g., U.S. Pat. No. 7,842,663, that,surprisingly, an ActRIIB construct beginning at 22, 23, 24, or 25 willhave the most activity.

Taken together, a general formula for an active portion (e.g.,ligand-binding portion) of ActRIIB comprises amino acids 29-109 of SEQID NO: 1. Therefore ActRIIB polypeptides may, for example, comprise,consist essentially of, or consist of an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIBbeginning at a residue corresponding to any one of amino acids 20-29(e.g., beginning at any one of amino acids 20, 21, 22, 23, 24, 25, 26,27, 28, or 29) of SEQ ID NO: 1 and ending at a position corresponding toany one amino acids 109-134 (e.g., ending at any one of amino acids 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ IDNO: 1. Other examples include polypeptides that begin at a position from20-29 (e.g., any one of positions 20, 21, 22, 23, 24, 25, 26, 27, 28, or29) or 21-29 (e.g., any one of positions 21, 22, 23, 24, 25, 26, 27, 28,or 29) of SEQ ID NO: 1 and end at a position from 119-134 (e.g., any oneof positions 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, or 134), 119-133 (e.g., any one of positions 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133),129-134 (e.g., any one of positions 129, 130, 131, 132, 133, or 134), or129-133 (e.g., any one of positions 129, 130, 131, 132, or 133) of SEQID NO: 1. Other examples include constructs that begin at a positionfrom 20-24 (e.g., any one of positions 20, 21, 22, 23, or 24), 21-24(e.g., any one of positions 21, 22, 23, or 24), or 22-25 (e.g., any oneof positions 22, 22, 23, or 25) of SEQ ID NO: 1 and end at a positionfrom 109-134 (e.g., any one of positions 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, or 134), 119-134 (e.g., any one of positions119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, or 134) or 129-134 (e.g., any one of positions 129, 130, 131, 132,133, or 134) of SEQ ID NO: 1. Variants within these ranges are alsocontemplated, particularly those having at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identity to the corresponding portion of SEQ ID NO: 1.

The variations described herein may be combined in various ways. In someembodiments, ActRIIB variants comprise no more than 1, 2, 5, 6, 7, 8, 9,10 or 15 conservative amino acid changes in the ligand-binding pocket,and zero, one, or more non-conservative alterations at positions 40, 53,55, 74, 79 and/or 82 in the ligand-binding pocket. Sites outside thebinding pocket, at which variability may be particularly well tolerated,include the amino and carboxy termini of the extracellular domain (asnoted above), and positions 42-46 and 65-73 (with respect to SEQ ID NO:1). An asparagine-to-alanine alteration at position 65 (N65A) actuallyimproves ligand binding in the A64 background, and is thus expected tohave no detrimental effect on ligand binding in the R64 background [U.S.Pat. No. 7,842,663]. This change probably eliminates glycosylation atN65 in the A64 background, thus demonstrating that a significant changein this region is likely to be tolerated. While an R64A change is poorlytolerated, R64K is well-tolerated, and thus another basic residue, suchas H may be tolerated at position 64 [U.S. Pat. No. 7,842,663].Additionally, the results of the mutagenesis program described in theart indicate that there are amino acid positions in ActRIIB that areoften beneficial to conserve. With respect to SEQ ID NO: 1, theseinclude position 80 (acidic or hydrophobic amino acid), position 78(hydrophobic, and particularly tryptophan), position 37 (acidic, andparticularly aspartic or glutamic acid), position 56 (basic amino acid),position 60 (hydrophobic amino acid, particularly phenylalanine ortyrosine). Thus, the disclosure provides a framework of amino acids thatmay be conserved in ActRIIB polypeptides. Other positions that may bedesirable to conserve are as follows: position 52 (acidic amino acid),position 55 (basic amino acid), position 81 (acidic), 98 (polar orcharged, particularly E, D, R or K), all with respect to SEQ ID NO: 1.

In certain embodiments, the disclosure relates to heteromultimers thatcomprise at least one ActRIIB polypeptide, which includes fragments,functional variants, and modified forms thereof. Preferably, ActRIIBpolypeptides for use in accordance with the disclosure are soluble(e.g., an extracellular domain of ActRIIB) In other preferredembodiments, ActRIIB polypeptides for use in accordance with thedisclosure bind to one or more TGF-beta superfamily ligands. Therefore,in some embodiments, ActRIIB polypeptides for use in accordance with thedisclosure inhibit (antagonize) activity (e.g., inhibition of Smadsignaling) of one or more TGF-beta superfamily ligands. In someembodiments, heteromultimers of the disclosure comprise at least oneActRIIB polypeptide that comprises, consists essentially of, or consistsof an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a portion of ActRIIB beginning at a residue correspondingto amino acids 20-29 (e.g., beginning at any one of amino acids 20, 21,22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 and ending at aposition corresponding to amino acids 109-134 (e.g., ending at any oneof amino acids 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or134) of SEQ ID NO: 1. In certain preferred embodiments, heteromultimersof the disclosure comprise at least one ActRIIB polypeptide thatcomprises, consists, or consists essentially of an amino acid sequencethat is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids 29-109of SEQ ID NO: 1. In other preferred embodiments, heteromultimers of thedisclosure comprise at least one ActRIIB polypeptide that comprises,consists, or consists essentially of an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical amino acids 25-131 of SEQ IDNO: 1. In some embodiments, heteromultimers of the disclosure compriseat least one ActRIIB polypeptide that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100%identical to the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3,4, 5, 6, 71, 73, 77, and 78. In certain embodiments, heteromultimers ofthe disclosure comprise at least one ActRIIB polypeptide wherein theamino acid position corresponding to L79 of SEQ ID NO: 1 is not anacidic amino acid (i.e., is not a naturally occurring D or E amino acidresidue or artificial acidic amino acid).

In certain aspects, the present disclosure relates to protein complexesthat comprise an ALK7 polypeptide. As used herein, the term “ALK7”refers to a family of activin receptor-like kinase-7 proteins from anyspecies and variants derived from such ALK7 proteins by mutagenesis orother modification. Reference to ALK7 herein is understood to be areference to any one of the currently identified forms. Members of theALK7 family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ALK7 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ALK7 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all ALK7-related polypeptides described herein is based on thenumbering of the human ALK7 precursor protein sequence below (SEQ ID NO:9), unless specifically designated otherwise.

There are various naturally occurring isoforms of human ALK7. Thesequence of canonical human ALK7 isoform 1 precursor protein (NCBI RefSeq NP_660302.2) is as follows:

(SEQ ID NO: 9) 1 MTRALCSALR QALLLLAAAA ELSPGLKCVC LLCDSSNFTCQTEGACWASV MLTNGKEQVI  61 KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLPTASPNAPKLG PMELAIIITV  121 PVCLLSIAAM LTVWACQGRQ CSYRKKKRPN VEEPLSECNLVNAGKTLKDL IYDVTASGSG  181 SGLPLLVQRT IARTIVLQEI VGKGRFGEVW HGRWCGEDVAVKIFSSRDER SWFREAEIYQ  241 TVMLRHENIL GFIAADNKDN GTWTQLWLVS EYHEQGSLYDYLNRNIVTVA GMIKLALSIA  301 SGLAHLHMEI VGTQGKPAIA HRDIKSKNIL VKKCETCAIADLGLAVKHDS ILNTIDIPQN  361 PKVGTKRYMA PEMLDDTMNV NIFESFKRAD IYSVGLVYWEIARRCSVGGI VEEYQLPYYD  421 MVPSDPSIEE MRKVVCDQKF RPSIPNQWQS CEALRVMGRIMRECWYANGA ARLTALRIKK  481 TISQLCVKED CKA

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

The processed extracellular ALK7 isoform 1 polypeptide sequence is asfollows:

(SEQ ID NO: 10) ELSPGLKCVCLLCDSSNFTCQTEGACWASVMLTNGKEQVIKSCVSLPELNAQVFCHSSNNVTKTECCFTDFCNNITLHLPTASPNAPKLGPME

A nucleic acid sequence encoding human ALK7 isoform 1 precursor proteinis shown below (SEQ ID NO: 11), corresponding to nucleotides 244-1722 ofGenbank Reference Sequence NM_145259.2. The signal sequence isunderlined and the extracellular domain is indicated in bold font.

(SEQ ID NO: 11) ATGACCCGGGCGCTCTGCTCAGCGCTCCGCCAGGCTCTCCTGCTGCTCGCAGCGGCCGCC GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGCATCACCAAATGCCCCAAAACTTGGACCCATGGAGCTGGCCATCATTATTACTGTGCCTGTTTGCCTCCTGTCCATAGCTGCGATGCTGACAGTATGGGCATGCCAGGGTCGACAGTGCTCCTACAGGAAGAAAAAGAGACCAAATGTGGAGGAACCACTCTCTGAGTGCAATCTGGTAAATGCTGGAAAAACTCTGAAAGATCTGATTTATGATGTGACCGCCTCTGGATCTGGCTCTGGTCTACCTCTGTTGGTTCAAAGGACAATTGCAAGGACGATTGTGCTTCAGGAAATAGTAGGAAAAGGTAGATTTGGTGAGGTGTGGCATGGAAGATGGTGTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGAAAGATCTTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACATGAAAACATCCTTGGTTTCATTGCTGCTGACAACAAAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACTGCAAAGCC

A nucleic acid sequence encoding the processed extracellular ALK7polypeptide (isoform 1) is as follows:

(SEQ ID NO: 12) GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGCATCACCAAATGCCCCAAAACTTGGACCCATGGAG

The amino acid sequence of an alternative isoform of human ALK7, isoform2 (NCBI Ref Seq NP_001104501.1), is shown in its processed form asfollows (SEQ ID NO: 19), where the extracellular domain is indicated inbold font.

(SEQ ID NO: 19) 1 MLTNGKEQVI KSCVSLPELN AQVFCHSSNN VTKTECCFTDFCNNITLHLP TASPNAPKLG  61 PMELAIIITV PVCLLSIAAM LTVWACQGRQ CSYRKKKRPNVEEPLSECNL VNAGKTLKDL  121 IYDVTASGSG SGLPLLVQRT IARTIVLQEI VGKGRFGEVWHGRWCGEDVA VKIFSSRDER  181 SWFREAEIYQ TVMLRHENIL GFIAADNKDN GTWTQLWLVSEYHEQGSLYD YLNRNIVTVA  241 GMIKLALSIA SGLAHLHMEI VGTQGKPAIA HRDIKSKNILVKKCETCAIA DLGLAVKHDS  301 ILNTIDIPQN PKVGTKRYMA PEMLDDTMNV NIFESFKRADIYSVGLVYWE IARRCSVGGI  361 VEEYQLPYYD MVPSDPSIEE MRKVVCDQKF RPSIPNQWQSCEALRVMGRI MRECWYANGA  421 ARLTALRIKK TISQLCVKED CKA 

The amino acid sequence of the extracellular ALK7 polypeptide (isoform2) is as follows:MLTNGKEQVIKSCVSLPELNAQVFCHSSNNVTKTECCFTDFCNNITLHLPTASPNAPKLGPME (SEQ IDNO: 20).

A nucleic acid sequence encoding the processed ALK7 polypeptide (isoform2) is shown below (SEQ ID NO: 21), corresponding to nucleotides 279-1607of NCBI Reference Sequence NM_001111031.1. The extracellular domain isindicated in bold font.

(SEQ ID NO: 21) ATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGCATCACCAAATGCCCCAAAACTTGGACCCATGGAGCTGGCCATCATTATTACTGTGCCTGTTTGCCTCCTGTCCATAGCTGCGATGCTGACAGTATGGGCATGCCAGGGTCGACAGTGCTCCTACAGGAAGAAAAAGAGACCAAATGTGGAGGAACCACTCTCTGAGTGCAATCTGGTAAATGCTGGAAAAACTCTGAAAGATCTGATTTATGATGTGACCGCCTCTGGATCTGGCTCTGGTCTACCTCTGTTGGTTCAAAGGACAATTGCAAGGACGATTGTGCTTCAGGAAATAGTAGGAAAAGGTAGATTTGGTGAGGTGTGGCATGGAAGATGGTGTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGAAAGATCTTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACATGAAAACATCCTTGGTTTCATTGCTGCTGACAACAAAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACTGCAAAGCC

A nucleic acid sequence encoding the extracellular ALK7 polypeptide(isoform 2) is as follows (SEQ ID NO: 22):

(SEQ ID NO: 22) ATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGCATCACCAAATGCCCCAAAACTTGGACCCATGGAG

The amino acid sequence of an alternative human ALK7 precursor protein,isoform 3 (NCBI Ref Seq NP_001104502.1), is shown as follows (SEQ ID NO:38), where the signal peptide is indicated by a single underline.

(SEQ ID NO: 38) 1 MTRALCSALR QALLLLAAAA ELSPGLKCVC LLCDSSNFTCQTEGACWASV MLTNGKEQVI  61 KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLPTGLPLLVQRT IARTIVLQEI  121 VGKGRFGEVW HGRWCGEDVA VKIFSSRDER SWFREAEIYQTVMLRHENIL GFIAADNKDN  181 GTWTQLWLVS EYHEQGSLYD YLNRNIVTVA GMIKLALSIASGLAHLHMEI VGTQGKPAIA  241 HRDIKSKNIL VKKCETCAIA DLGLAVKHDS ILNTIDIPQNPKVGTKRYMA PEMLDDTMNV  301 NIFESFKRAD IYSVGLVYWE IARRCSVGGI VEEYQLPYYDMVPSDPSIEE MRKVVCDQKF  361 RPSIPNQWQS CEALRVMGRI MRECWYANGA ARLTALRIKKTISQLCVKED CKA 

The amino acid sequence of the processed ALK7 polypeptide (isoform 3) isas follows (SEQ ID NO: 39). This isoform lacks a transmembrane domainand is therefore proposed to be soluble in its entirety (Roberts et al.,2003, Biol Reprod 68:1719-1726). N-terminal variants of SEQ ID NO: 39are predicted as described below.

(SEQ ID NO: 39) 1 ELSPGLKCVC LLCDSSNFTC QTEGACWASV MLTNGKEQVIKSCVSLPELN AQVFCHSSNN  61 VTKTECCFTD FCNNITLHLP TGLPLLVQRT IARTIVLQEIVGKGRFGEVW HGRWCGEDVA  121 VKIFSSRDER SWFREAEIYQ TVMLRHENIL GFIAADNKDNGTWTQLWLVS EYHEQGSLYD  181 YLNRNIVTVA GMIKLALSIA SGLAHLHMEI VGTQGKPAIAHRDIKSKNIL VKKCETCAIA  241 DLGLAVKHDS ILNTIDIPQN PKVGTKRYMA PEMLDDTMNVNIFESFKRAD IYSVGLVYWE  301 IARRCSVGGI VEEYQLPYYD MVPSDPSIEE MRKVVCDQKFRPSIPNQWQS CEALRVMGRI  361 MRECWYANGA ARLTALRIKK TISQLCVKED CKA

A nucleic acid sequence encoding the unprocessed ALK7 polypeptideprecursor protein (isoform 3) is shown below (SEQ ID NO: 40),corresponding to nucleotides 244-1482 of NCBI Reference SequenceNM_001111032.1. The signal sequence is indicated by solid underline.

(SEQ ID NO: 40) ATGACCCGGGCGCTCTGCTCAGCGCTCCGCCAGGCTCTCCTGCTGCTCGCAGCGGCCGCCGAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGGTCTACCTCTGTTGGTTCAAAGGACAATTGCAAGGACGATTGTGCTTCAGGAAATAGTAGGAAAAGGTAGATTTGGTGAGGTGTGGCATGGAAGATGGTGTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGAAAGATCTTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACATGAAAACATCCTTGGTTTCATTGCTGCTGACAACAAAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAG AAGACTGCAAAGCC

A nucleic acid sequence encoding the processed ALK7 polypeptide (isoform3) is as follows (SEQ ID NO: 41):

(SEQ ID NO: 41) GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGGTCTACCTCTGTTGGTTCAAAGGACAATTGCAAGGACGATTGTGCTTCAGGAAATAGTAGGAAAAGGTAGATTTGGTGAGGTGTGGCATGGAAGATGGTGTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGAAAGATCTTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACATGAAAACATCCTTGGTTTCATTGCTGCTGACAACAAAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACTGCAAA GCC

The amino acid sequence of an alternative human ALK7 precursor protein,isoform 4 (NCBI Ref Seq NP_001104503.1), is shown as follows (SEQ ID NO:42), where the signal peptide is indicated by a single underline.

(SEQ ID NO: 42)   1MTRALCSALR QALLLLAAAA ELSPGLKCVC LLCDSSNFTC QTEGACWASV MLTNGKEQVI  61KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLP TDNGTWTQLW LVSEYHEQGS 121LYDYLNRNIV TVAGMIKLAL SIASGLAHLH MEIVGTQGKP AIAHRDIKSK NILVKKCETC 181AIADLGLAVK HDSILNTIDI PQNPKVGTKR YMAPEMLDDT MNVNIFESFK RADIYSVGLV 241YWEIARRCSV GGIVEEYQLP YYDMVPSDPS IEEMRKVVCD QKFRPSIPNQ WQSCEALRVM 301GRIMRECWYA NGAARLTALR IKKTISQLCV KEDCKA

The amino acid sequence of the processed ALK7 polypeptide (isoform 4) isas follows (SEQ ID NO: 43). Like ALK7 isoform 3, isoform 4 lacks atransmembrane domain and is therefore proposed to be soluble in itsentirety (Roberts et al., 2003, Biol Reprod 68:1719-1726). N-terminalvariants of SEQ ID NO: 43 are predicted as described below.

(SEQ ID NO: 43)   1ELSPGLKCVC LLCDSSNFTC QTEGACWASV MLTNGKEQVI KSCVSLPELN AQVFCHSSNN  61VTKTECCFTD FCNNITLHLP TDNGTWTQLW LVSEYHEQGS LYDYLNRNIV TVAGMIKLAL 121SIASGLAHLH MEIVGTQGKP AIAHRDIKSK NILVKKCETC AIADLGLAVK HDSILNTIDI 181PQNPKVGTKR YMAPEMLDDT MNVNIFESFK RADIYSVGLV YWEIARRCSV GGIVEEYQLP 240YYDMVPSDPS IEEMRKVVCD QKFRPSIPNQ WQSCEALRVM GRIMRECWYA NGAARLTALR 301IKKTISQLCV KEDCKA

A nucleic acid sequence encoding the unprocessed ALK7 polypeptideprecursor protein (isoform 4) is shown below (SEQ ID NO: 44),corresponding to nucleotides 244-1244 of NCBI Reference SequenceNM_001111033.1. The signal sequence is indicated by solid underline.

(SEQ ID NO: 44) ATGACCCGGGCGCTCTGCTCAGCGCTCCGCCAGGCTCTCCTGCTGCTCGCAGCGGCCGCCGAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACTGCAAAGCCTAA

A nucleic acid sequence encoding the processed ALK7 polypeptide (isoform4) is as follows (SEQ ID NO: 45):

(SEQ ID NO: 45) GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCA AAGAAGACTGCAAAGCCTAA

Based on the signal sequence of full-length ALK7 (isoform 1) in the rat(see NCBI Reference Sequence NP_620790.1) and on the high degree ofsequence identity between human and rat ALK7, it is predicted that aprocessed form of human ALK7 isoform 1 is as follows (SEQ ID NO: 46).

1 LKCVCLLCDS SNFTCQTEGA CWASVMLTNG KEQVIKSCVS LPELNAQVFC HSSNNVTKTE

61 CCFTDFCNNI TLHLPTASPN APKLGPME (SEQ ID NO: 46)

Active variants of processed ALK7 isoform 1 are predicted in which SEQID NO: 10 is truncated by 1, 2, 3, 4, 5, 6, or 7 amino acids at theN-terminus and SEQ ID NO: 46 is truncated by 1 or 2 amino acids at theN-terminus. Consistent with SEQ ID NO: 46, it is further expected thatleucine is the N-terminal amino acid in the processed forms of humanALK7 isoform 3 (SEQ ID NO: 39) and human ALK7 isoform 4 (SEQ ID NO: 43).

In certain embodiments, the disclosure relates to heteromultimers thatcomprise at least one ALK7 polypeptide, which includes fragments,functional variants, and modified forms thereof. Preferably, ALK7polypeptides for use in accordance with inventions of the disclosure(e.g., heteromultimers comprising an ALK7 polypeptide and uses thereof)are soluble (e.g., an extracellular domain of ALK7). In other preferredembodiments, ALK7 polypeptides for use in accordance with the disclosurebind to one or more TGF-beta superfamily ligands. Therefore, in somepreferred embodiments, ALK7 polypeptides for use in accordance with thedisclosure inhibit (antagonize) activity (e.g., induction of Smad 2/3and/or Smad 1/5/8 signaling) of one or more TGF-beta superfamilyligands. In some embodiments, heteromultimers of the disclosure compriseat least one ALK7 polypeptide that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identicalto the amino acid sequence of SEQ ID NO: 9, 10, 19, 20, 38, 39, 42, 43,46, 74, 75, 79, or 80. In some embodiments, heteromultimer of thedisclosure consist or consist essentially of at least one ALK7polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identical to the aminoacid sequence of SEQ ID NO: 9, 10, 19, 20, 38, 39, 42, 43, 46, 74, 75,79, and 80.

ALK7 is well-conserved among vertebrates, with large stretches of theextracellular domain completely conserved. For example, FIG. 7 depicts amulti-sequence alignment of a human ALK7 extracellular domain comparedto various ALK7 orthologs. Accordingly, from these alignments, it ispossible to predict key amino acid positions within the ligand-bindingdomain that are important for normal ALK7-ligand binding activities aswell as to predict amino acid positions that are likely to be tolerantto substitution without significantly altering normal ALK7-ligandbinding activities. Therefore, an active, human ALK7 variant polypeptideuseful in accordance with the presently disclosed methods may includeone or more amino acids at corresponding positions from the sequence ofanother vertebrate ALK7, or may include a residue that is similar tothat in the human or other vertebrate sequences. Without meaning to belimiting, the following examples illustrate this approach to defining anactive ALK7 variant. V61 in the human ALK7 extracellular domain (SEQ IDNO: 59) is isoleucine in Callithrix jacchus ALK7 (SEQ ID NO: 62), and sothe position may be altered, and optionally may be altered to anotherhydrophobic residue such as L, I, or F, or a non-polar residue such asA. L32 in the human extracellular domain is R in Tarsius syrichta (SEQID NO: 61) ALK7, indicating that this site may be tolerant of a widevariety of changes, including polar residues, such as E, D, K, R, H, S,T, P, G, Y, and probably a non-polar residue such as A. K37 in the humanextracellular domain is R in Pan troglodytes ALK7 (SEQ ID NO: 60),indicating that basic residues are tolerated at this position, includingR, K, and H. P4 in the human extracellular domain is relatively poorlyconserved, appearing as A in Pan troglodytes ALK7 thus indicating that awide variety of amino acid should be tolerated at this position.

Moreover, ALK7 proteins have been characterized in the art in terms ofstructural and functional characteristics [e.g., Romano et al (2012)Journal of Molecular Modeling 18(8): 3617-3625]. For example, a definingstructural motif known as a three-finger toxin fold is important forligand binding by type I and type II receptors and is formed byconserved cysteine residues located at varying positions within theextracellular domain of each monomeric receptor [Greenwald et al. (1999)Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870].Accordingly, the core ligand-binding domains of human ALK7, asdemarcated by the outermost of these conserved cysteines, corresponds topositions 28-92 of SEQ ID NO: 9. The structurally less-ordered aminoacids flanking these cysteine-demarcated core sequences can be truncatedby 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, or 27 residues at the N-terminus and by 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or21 residues at the C-terminus without necessarily altering ligandbinding. Exemplary ALK7 extracellular domains for N-terminal and/orC-terminal truncation include SEQ ID NOs: 10, 20, 39, and 43.

Accordingly, a general formula for an active portion (e.g., aligand-binding portion) of ALK7 comprises amino acids 28-92. ThereforeALK7 polypeptides may, for example, comprise, consists essentially of,or consists of an amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to a portion of ALK7 beginning at a residuecorresponding to any one of amino acids 20-28 (e.g., beginning at anyone of amino acids 20, 21, 22, 23, 24, 25, 26, 27, or 28) of SEQ ID NO:9 and ending at a position corresponding to any one amino acids 92-113(e.g., ending at any one of amino acids 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113)of SEQ ID NO: 9.

Other examples include constructs that begin at a position from 21-28(e.g., any one of positions 21, 22, 23, 24, 25, 26, 27, or 28), 24-28(e.g., any one of positions 24, 25, 26, 27, or 28), or 25-28 (e.g., anyone of positions 25, 26, 27, or 28) of SEQ ID NO: 9 and end at aposition from 93-112 (e.g., any one of positions 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112),93-110 (e.g., any one of positions 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, or 110), 93-100 (e.g., any oneof positions 93, 94, 95, 96, 97, 98, 99, or 100), or 93-95 (e.g., anyone of positions 93, 94, or 95) of SEQ ID NO: 9. Variants within theseranges are also contemplated, particularly those having at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to the corresponding portion of SEQ IDNO: 9.

The variations described herein may be combined in various ways. In someembodiments, ALK7 variants comprise no more than 1, 2, 5, 6, 7, 8, 9, 10or 15 conservative amino acid changes in the ligand-binding pocket.Sites outside the binding pocket, at which variability may beparticularly well tolerated, include the amino and carboxy termini ofthe extracellular domain (as noted above).

In certain aspects, the present disclosure relates to heteromultimerscomprising one or more ALK7 receptor polypeptides (e.g., SEQ ID Nos: 9,10, 19, 20, 38, 39, 42, 43, 46, 74, 76, 79, and 80) and one or moreActRIIB receptor polypeptides (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 71,73, 77, and 78), which are generally referred to herein as “ALK7:ActRIIBheteromultimer complexes” or “ALK7:ActRIIB heteromultimers”. Preferably,ALK7:ActRIIB heteromultimers are soluble, e.g., a heteromultimercomprises a soluble portion (domain) of an ALK7 receptor and a solubleportion (domain) of an ActRIIB receptor. In general, the extracellulardomains of ALK7 and ActRIIB correspond to soluble portion of thesereceptors. Therefore, in some embodiments, heteromultimers comprise anextracellular domain of an ALK7 receptor and an extracellular domain ofan ActRIIB receptor. Example extracellular domains of ALK7 and ActRIIBreceptors are disclosed herein and such sequences, as well as fragments,functional variants, and modified forms thereof, may be used inaccordance with the inventions of the disclosure (e.g., ALK7:ActRIIBheteromultimer compositions and uses thereof). In some embodiments,ALK7:ActRIIB heteromultimers comprise at least one ALK7 polypeptide thatcomprises, consists essentially of, or consists of a sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQID NO: 9, 10, 19, 20, 38, 39, 42, 43, 46, 74, 76, 79, and 80. In someembodiments, ALK7:ActRIIB heteromultimers comprise at least one ALK7polypeptide that comprises, consists essentially of, consists of asequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94% 95%, 97%, 98%, 99%, or 100% identical to a portion ofALK7 beginning at a residue corresponding to any one of amino acids21-28, 22-28, 23-28, 24-28, 25-28, 26-28, or 27-28 of SEQ ID NO: 9 andending at a position from 93-112, 93, 110, 93-100, 93-95 of SEQ ID NO:9. In some embodiments, ALK7:ActRIIB heteromultimers comprise at leastone ALK7 polypeptide that comprises, consists essentially of, consistsof a sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94% 95%, 97%, 98%, 99%, or 100% identical to aminoacids 28-92 of SEQ ID NO: 9. In some embodiments, ALK7-ActRIIBheteromultimers comprise at least one ActRIIB polypeptide thatcomprises, consists essentially of, consists of a sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of anyone of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 71, 73, 77, and 78. In someembodiments, ALK7:ActRIIB heteromultimers comprise at least one ActRIIBpolypeptide that comprises, consists essentially of, consists of asequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94% 95%, 97%, 98%, 99%, or 100% identical to a portion ofActRIIB beginning at a residue corresponding to any one of amino acids20-29, 20-24, 21-24, 22-25, or 21-29 and end at a position from 109-134,119-134, 119-133, 129-134, or 129-133 of SEQ ID NO: 1. In someembodiments, ALK7:ActRIIB heteromultimers comprise at least one ActRIIBpolypeptide that comprises, consists essentially of, or consists of asequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94% 95%, 97%, 98%, 99%, or 100% identical to amino acids29-109 of SEQ ID NO: 1. In some embodiments, ALK7:ActRIIBheteromultimers comprise at least one ActRIIB polypeptide thatcomprises, consists essentially of, consists of a sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%95%, 97%, 98%, 99%, or 100% identical to amino acids 25-125 of SEQ IDNO: 1. In certain preferred embodiments, ALK7:ActRIIB heteromultimerscomprise at least one ActRIIB polypeptide wherein the positioncorresponding to L79 of SEQ ID NO: 1 is not an acidic amino acid (i.e.,not naturally occurring D or E amino acids or an artificial acidic acidresidue). ALK7:ActRIIB heteromultimers include, e.g., heterodimers,heterotrimers, heterotetramers and further higher order oligomericstructures. See, e.g., FIGS. 1, 2, and 8-10 . In certain preferredembodiments, heteromultimers of the disclosure are ALK7:ActRIIBheterodimers.

In some embodiments, the present disclosure contemplates makingfunctional variants by modifying the structure of an ALK7 polypeptideand/or an ActRIIB polypeptide. Variants can be produced by amino acidsubstitution, deletion, addition, or combinations thereof. For instance,it is reasonable to expect that an isolated replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar replacement of an amino acid with astructurally related amino acid (e.g., conservative mutations) will nothave a major effect on the biological activity of the resultingmolecule. Conservative replacements are those that take place within afamily of amino acids that are related in their side chains. Whether achange in the amino acid sequence of a polypeptide of the disclosureresults in a functional homolog can be readily determined by assessingthe ability of the variant polypeptide to produce a response in cells ina fashion similar to the wild-type polypeptide, or to bind to one ormore TGF-beta superfamily ligands including, for example, BMP2, BMP2/7,BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3,GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1,TGF-β1, TGF-β2, TGF-β3, activin A, activin B, activin C, activin E,activin AB, activin AC, activin AE, activin BC, activin BE, nodal, glialcell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin,MIS, and Lefty.

In some embodiments, the present disclosure contemplates makingfunctional variants by modifying the structure of an ALK7 polypeptideand/or an ActRIIB polypeptide for such purposes as enhancing therapeuticefficacy or stability (e.g., shelf-life and resistance to proteolyticdegradation in vivo).

In some embodiments, the present disclosure contemplates specificmutations of an ALK7 polypeptide and/or an ActRIIB polypeptide so as toalter the glycosylation of the polypeptide. Such mutations may beselected so as to introduce or eliminate one or more glycosylationsites, such as O-linked or N-linked glycosylation sites.Asparagine-linked glycosylation recognition sites generally comprise atripeptide sequence, asparagine-X-threonine or asparagine-X-serine(where “X” is any amino acid) which is specifically recognized byappropriate cellular glycosylation enzymes. The alteration may also bemade by the addition of, or substitution by, one or more serine orthreonine residues to the sequence of the polypeptide (for O-linkedglycosylation sites). A variety of amino acid substitutions or deletionsat one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Another means of increasing the number of carbohydratemoieties on a polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Depending on the coupling mode used, thesugar(s) may be attached to (a) arginine and histidine; (b) freecarboxyl groups; (c) free sulfhydryl groups such as those of cysteine;(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal ofone or more carbohydrate moieties present on a polypeptide may beaccomplished chemically and/or enzymatically. Chemical deglycosylationmay involve, for example, exposure of a polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. [Meth. Enzymol. (1987)138:350]. The sequence of a polypeptide may be adjusted, as appropriate,depending on the type of expression system used, as mammalian, yeast,insect, and plant cells may all introduce differing glycosylationpatterns that can be affected by the amino acid sequence of the peptide.In general, heteromeric complexes of the present disclosure for use inhumans may be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

The present disclosure further contemplates a method of generatingmutants, particularly sets of combinatorial mutants of an ALK7 and/or anActRIIB polypeptide as well as truncation mutants. Pools ofcombinatorial mutants are especially useful for identifying functionallyactive (e.g., TGF-beta superfamily ligand binding) ALK7 and/or ActRIIBsequences. The purpose of screening such combinatorial libraries may beto generate, for example, polypeptides variants which have alteredproperties, such as altered pharmacokinetic or altered ligand binding. Avariety of screening assays are provided below, and such assays may beused to evaluate variants. For example, ALK7:ActRIIB complex variantsmay be screened for ability to bind to one or more TGF-beta superfamilyligands, to prevent binding of a TGF-beta superfamily ligand to aTGF-beta superfamily receptor, and/or to interfere with signaling causedby an TGF-beta superfamily ligand.

The activity of a ALK7:ActRIIB heteromultimer may be tested, forexample, in a cell-based or in vivo assay. For example, the effect of anALK7:ActRIIB heteromultimer on the expression of genes or the activityof proteins involved in fat metabolism, kidney damage, kidneyinflammation, and/or kidney fibrosis may be assessed. This may, asneeded, be performed in the presence of one or more recombinant TGF-betasuperfamily ligand proteins, and cells may be transfected so as toproduce an ALK7:ActRIIB heteromultimer, and optionally, a TGF-betasuperfamily ligand. Likewise, an ALK7:ActRIIB heteromultimer may beadministered to a mouse or other animal, and one or more measurements,such as fat metabolism or kidney damage, inflammation, fibrosis and/orfunction may be assessed using art-recognized methods. Similarly, theactivity of an ALK7:ActRIIB heteromultimer, or variants thereof, may betested in, for example, adipocytes and/or glomerular cells for anyeffect on growth of these cells, for example, by the assays as describedherein and those of common knowledge in the art. A SMAD-responsivereporter gene may be used in such cell lines to monitor effects ondownstream signaling.

Combinatorial-derived variants can be generated which have increasedselectivity or generally increased potency relative to a referenceALK7:ActRIIB heteromultimer. Such variants, when expressed fromrecombinant DNA constructs, can be used in gene therapy protocols.Likewise, mutagenesis can give rise to variants which have intracellularhalf-lives dramatically different than the corresponding unmodifiedALK7:ActRIIB heteromultimer. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother cellular processes which result in destruction, or otherwiseinactivation, of an unmodified polypeptide. Such variants, and the geneswhich encode them, can be utilized to alter polypeptide complex levelsby modulating the half-life of the polypeptide. For instance, a shorthalf-life can give rise to more transient biological effects and, whenpart of an inducible expression system, can allow tighter control ofrecombinant polypeptide complex levels within the cell. In an Fc fusionprotein, mutations may be made in the linker (if any) and/or the Fcportion to alter one or more activities of the ALK7:ActRIIBheteromultimer including, for example, immunogenicity, half-life, andsolubility.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential ALK7 and/or ActRIIB sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential ALK7 and/orActRIIB encoding nucleotide sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes can then be ligated into anappropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art [Narang, S A (1983)Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rdCleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; and Ike et al. (1983) Nucleic Acid Res.11:477]. Such techniques have been employed in the directed evolution ofother proteins [Scott et al., (1990) Science 249:386-390; Roberts et al.(1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249: 404-406;Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos.5,223,409, 5,198,346, and 5,096,815].

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ALK7:ActRIIB heteromultimers can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis [Ruf et al. (1994) Biochemistry33:1565-1572; Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint etal. (1993) Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem.218:597-601; Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892;Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al.(1989) Science 244:1081-1085], by linker scanning mutagenesis [Gustin etal. (1993) Virology 193:653-660; and Brown et al. (1992) Mol. Cell Biol.12:2644-2652; McKnight et al. (1982) Science 232:316], by saturationmutagenesis [Meyers et al., (1986) Science 232:613]; by PCR mutagenesis[Leung et al. (1989) Method Cell Mol Biol 1:11-19]; or by randommutagenesis, including chemical mutagenesis [Miller et al. (1992) AShort Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY;and Greener et al. (1994) Strategies in Mol Biol 7:32-34]. Linkerscanning mutagenesis, particularly in a combinatorial setting, is anattractive method for identifying truncated (bioactive) forms of ALK7and/or ActRIIB polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ALK7:ActRIIB heteromultimers. The mostwidely used techniques for screening large gene libraries typicallycomprise cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferredassays include TGF-beta superfamily ligand (e.g., BMP2, BMP2/7, BMP3,BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5,GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1,TGF-β2, TGF-β3, activin A, activin B, activin C, activin E, activin AB,activin AC, activin BC, activin AE, activin BE, nodal, glialcell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin,MIS, and Lefty) binding assays and/or TGF-beta superfamilyligand-mediated cell signaling assays.

In certain embodiments, ALK7:ActRIIB heteromultimers may furthercomprise post-translational modifications in addition to any that arenaturally present in the ALK7 and/or ActRIIB polypeptide. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, ALK7:ActRIIB heteromultimers may comprisenon-amino acid elements, such as polyethylene glycols, lipids,polysaccharide or monosaccharide, and phosphates. Effects of suchnon-amino acid elements on the functionality of a heteromultimer complexmay be tested as described herein for other heteromultimer variants.When a polypeptide of the disclosure is produced in cells by cleaving anascent form of the polypeptide, post-translational processing may alsobe important for correct folding and/or function of the protein.Different cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293)have specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the ALK7 and/or ActRIIB polypeptide aswell as heteromultimers comprising the same.

In certain preferred embodiments, heteromultimers described hereincomprise at least one ALK7 polypeptide associated, covalently ornon-covalently, with at least one ActRIIB polypeptide. Preferably,polypeptides disclosed herein form heterodimeric complexes, althoughhigher order heteromultimeric complexes are also included such as, butnot limited to, heterotrimers, heterotetramers, and further oligomericstructures (see, e.g., FIGS. 1, 2, and 8-10 ). In some embodiments, ALK7and/or ActRIIB polypeptides comprise at least one multimerizationdomain. As disclosed herein, the term “multimerization domain” refers toan amino acid or sequence of amino acids that promote covalent ornon-covalent interaction between at least a first polypeptide and atleast a second polypeptide. Polypeptides disclosed herein may be joinedcovalently or non-covalently to a multimerization domain. Preferably, amultimerization domain promotes interaction between a first polypeptide(e.g., an ALK7 polypeptide) and a second polypeptide (e.g., an ActRIIBpolypeptide) to promote heteromultimer formation (e.g., heterodimerformation), and optionally hinders or otherwise disfavors homomultimerformation (e.g., homodimer formation), thereby increasing the yield ofdesired heteromultimer (see, e.g., FIG. 2 ).

Many methods known in the art can be used to generate ALK7:ActRIIBheteromultimers. For example, non-naturally occurring disulfide bondsmay be constructed by replacing on a first polypeptide (e.g., an ALK7polypeptide) a naturally occurring amino acid with a freethiol-containing residue, such as cysteine, such that the free thiolinteracts with another free thiol-containing residue on a secondpolypeptide (e.g., an ActRIIB polypeptide) such that a disulfide bond isformed between the first and second polypeptides. Additional examples ofinteractions to promote heteromultimer formation include, but are notlimited to, ionic interactions such as described in Kjaergaard et al.,WO2007147901; electrostatic steering effects such as described in Kannanet al., U.S. Pat. No. 8,592,562; coiled-coil interactions such asdescribed in Christensen et al., U.S. 20120302737; leucine zippers suchas described in Pack & Plueckthun, (1992) Biochemistry 31: 1579-1584;and helix-turn-helix motifs such as described in Pack et al., (1993)Bio/Technology 11: 1271-1277. Linkage of the various segments may beobtained via, e.g., covalent binding such as by chemical cross-linking,peptide linkers, disulfide bridges, etc., or affinity interactions suchas by avidin-biotin or leucine zipper technology.

In certain aspects, a multimerization domain may comprise one componentof an interaction pair. In some embodiments, the polypeptides disclosedherein may form protein complexes comprising a first polypeptidecovalently or non-covalently associated with a second polypeptide,wherein the first polypeptide comprises the amino acid sequence of anALK7 polypeptide and the amino acid sequence of a first member of aninteraction pair; and the second polypeptide comprises the amino acidsequence of an ActRIIB polypeptide and the amino acid sequence of asecond member of an interaction pair. The interaction pair may be anytwo polypeptide sequences that interact to form a complex, particularlya heterodimeric complex although operative embodiments may also employan interaction pair that can form a homodimeric complex. One member ofthe interaction pair may be fused to an ALK7 or ActRIIB polypeptide asdescribed herein, including for example, a polypeptide sequencecomprising, consisting essentially of, or consisting of an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence of any one of SEQ ID NOs: 2, 3, 5, 6, 10, 20, 39, 43, and 46.An interaction pair may be selected to confer an improvedproperty/activity such as increased serum half-life, or to act as anadaptor on to which another moiety is attached to provide an improvedproperty/activity. For example, a polyethylene glycol moiety may beattached to one or both components of an interaction pair to provide animproved property/activity such as improved serum half-life.

The first and second members of the interaction pair may be anasymmetric pair, meaning that the members of the pair preferentiallyassociate with each other rather than self-associate. Accordingly, firstand second members of an asymmetric interaction pair may associate toform a heterodimeric complex (see, e.g., FIG. 2 ). Alternatively, theinteraction pair may be unguided, meaning that the members of the pairmay associate with each other or self-associate without substantialpreference and thus may have the same or different amino acid sequences.Accordingly, first and second members of an unguided interaction pairmay associate to form a homodimer complex or a heterodimeric complex.Optionally, the first member of the interaction pair (e.g., anasymmetric pair or an unguided interaction pair) associates covalentlywith the second member of the interaction pair. Optionally, the firstmember of the interaction pair (e.g., an asymmetric pair or an unguidedinteraction pair) associates non-covalently with the second member ofthe interaction pair.

As specific examples, the present disclosure provides fusion proteinscomprising ALK7 or ActRIIB fused to a polypeptide comprising a constantdomain of an immunoglobulin, such as a CH1, CH2, or CH3 domain of animmunoglobulin or an Fc domain. Fc domains derived from human IgG1,IgG2, IgG3, and IgG4 are provided herein. Other mutations are known thatdecrease either CDC or ADCC activity, and collectively, any of thesevariants are included in the disclosure and may be used as advantageouscomponents of a heteromultimeric complex of the disclosure. Optionally,the IgG1 Fc domain of SEQ ID NO: 31 has one or more mutations atresidues such as Asp-265, Lys-322, and Asn-434 (numbered in accordancewith the corresponding full-length IgG1). In certain cases, the mutantFc domain having one or more of these mutations (e.g., Asp-265 mutation)has reduced ability of binding to the Fcγ receptor relative to awildtype Fc domain. In other cases, the mutant Fc domain having one ormore of these mutations (e.g., Asn-434 mutation) has increased abilityof binding to the MHC class I-related Fc-receptor (FcRN) relative to awildtype Fc domain.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG1 (G1Fc) is shown below (SEQ ID NO: 31). Dottedunderline indicates the hinge region, and solid underline indicatespositions with naturally occurring variants. In part, the disclosureprovides polypeptides comprising, consisting essentially of, orconsisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity to SEQ ID NO: 31. Naturally occurring variants in G1Fc wouldinclude E134D and M136L according to the numbering system used in SEQ IDNO: 31 (see Uniprot P01857).

(SEQ ID NO: 31)   1

 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK

An example of a native amino acid sequence that may be used for the Fcportion of human IgG2 (G2Fc) is shown below (SEQ ID NO: 32). Dottedunderline indicates the hinge region and double underline indicatespositions where there are data base conflicts in the sequence (accordingto UniProt P01859). In part, the disclosure provides polypeptidescomprising, consisting essential of, or consisting of amino acidsequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 32.

(SEQ ID NO: 32)   1

 51 FNWYVDGVEV HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS 101NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP 151SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS 201CSVMHEALHN HYTQKSLSLS PGK

Two examples of amino acid sequences that may be used for the Fc portionof human IgG3 (G3Fc) are shown below. The hinge region in G3Fc can be upto four times as long as in other Fc chains and contains three identical15-residue segments preceded by a similar 17-residue segment. The firstG3Fc sequence shown below (SEQ ID NO: 33) contains a short hinge regionconsisting of a single 15-residue segment, whereas the second G3Fcsequence (SEQ ID NO: 34) contains a full-length hinge region. In eachcase, dotted underline indicates the hinge region, and solid underlineindicates positions with naturally occurring variants according toUniProt P01859. In part, the disclosure provides polypeptidescomprising, consisting essential of, or consisting of amino acidsequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 33and 34.

(SEQ ID NO: 33)   1

 51 VSHEDPEVQF KWYVDGVEVH NAKTKPREEQ YNSTFRVVSV LTVLHQDWLN 101GKEYKCKVSN KALPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL 151TCLVKGFYPS DIAVEWESSG QPENNYNTTP PMLDSDGSFF LYSKLTVDKS 201RWQQGNIFSC SVMHEALHNR FTQKSLSLSP GK (SEQ ID NO: 34)   1

 51

101 EDPEVQFKWY VDGVEVHNAK TKPREEQYNS TFRVVSVLTV LHQDWLNGKE 151YKCKVSNKAL PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL 201VKGFYPSDIA VEWESSGQPE NNYNTTPPML DSDGSFFLYS KLTVDKSRWQ 251QGNIFSCSVM HEALHNRFTQ KSLSLSPGK

Naturally occurring variants in G3Fc (for example, see Uniprot P01860)include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169del,F221Y when converted to the numbering system used in SEQ ID NO: 33, andthe present disclosure provides fusion proteins comprising G3Fc domainscontaining one or more of these variations. In addition, the humanimmunoglobulin IgG3 gene (IGHG3) shows a structural polymorphismcharacterized by different hinge lengths [see Uniprot P01859].Specifically, variant WIS is lacking most of the V region and all of theCH1 region. It has an extra interchain disulfide bond at position 7 inaddition to the 11 normally present in the hinge region. Variant ZUClacks most of the V region, all of the CH1 region, and part of thehinge. Variant OMM may represent an allelic form or another gamma chainsubclass. The present disclosure provides additional fusion proteinscomprising G3Fc domains containing one or more of these variants.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG4 (G4Fc) is shown below (SEQ ID NO: 35). Dottedunderline indicates the hinge region. In part, the disclosure providespolypeptides comprising, consisting essential of, or consisting of aminoacid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:35.

(SEQ ID NO: 35)   1

 51 EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE 101YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL 151VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ 201EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

A variety of engineered mutations in the Fc domain are presented hereinwith respect to the G1Fc sequence (SEQ ID NO: 31), and analogousmutations in G2Fc, G3Fc, and G4Fc can be derived from their alignmentwith G1Fc in FIG. 5 . Due to unequal hinge lengths, analogous Fcpositions based on isotype alignment (FIG. 5 ) possess different aminoacid numbers in SEQ ID NOs: 31, 32, 33, 34, and 35. It can also beappreciated that a given amino acid position in an immunoglobulinsequence consisting of hinge, C_(H)2, and C_(H)3 regions (e.g., SEQ IDNOs: 31, 32, 33, 34, and 35) will be identified by a different numberthan the same position when numbering encompasses the entire IgG1heavy-chain constant domain (consisting of the C_(H)1, hinge, C_(H)2,and C_(H)3 regions) as in the Uniprot database. For example,correspondence between selected C_(H)3 positions in a human G1Fcsequence (SEQ ID NO: 31), the human IgG1 heavy chain constant domain(Uniprot P01857), and the human IgG1 heavy chain is as follows.

Correspondence of C_(H)3 Positions in Different Numbering Systems G1Fc(Numbering begins IgG1 heavy chain IgG1 heavy chain at first constantdomain (EU numbering threonine in (Numbering begins scheme of hingeregion) at C_(H)1) Kabat et al., 1991*) Y127 Y232 Y349 S132 S237 S354E134 E239 E356 T144 T249 T366 L146 L251 L368 K170 K275 K392 D177 D282D399 Y185 Y290 Y407 K187 K292 K409 *Kabat et al. (eds) 1991; pp. 688-696in Sequences of Proteins of Immunological Interest, 5^(th) ed., Vol. 1,NIH, Bethesda, MD.

A problem that arises in large-scale production of asymmetricimmunoglobulin-based proteins from a single cell line is known as the“chain association issue”. As confronted prominently in the productionof bispecific antibodies, the chain association issue concerns thechallenge of efficiently producing a desired multichain protein fromamong the multiple combinations that inherently result when differentheavy chains and/or light chains are produced in a single cell line[Klein et al (2012) mAbs 4:653-663]. This problem is most acute when twodifferent heavy chains and two different light chains are produced inthe same cell, in which case there are a total of 16 possible chaincombinations (although some of these are identical) when only one istypically desired. Nevertheless, the same principle accounts fordiminished yield of a desired multichain fusion protein thatincorporates only two different (asymmetric) heavy chains.

Various methods are known in the art that increase desired pairing ofFc-containing fusion polypeptide chains in a single cell line to producea preferred asymmetric fusion protein at acceptable yields [Klein et al(2012) mAbs 4:653-663; and Spiess et al (2015) Molecular Immunology67(2A): 95-106]. Methods to obtain desired pairing of Fc-containingchains include, but are not limited to, charge-based pairing(electrostatic steering), “knobs-into-holes” steric pairing, SEEDbodypairing, and leucine zipper-based pairing [Ridgway et al (1996) ProteinEng 9:617-621; Merchant et al (1998) Nat Biotech 16:677-681; Davis et al(2010) Protein Eng Des Sel 23:195-202; Gunasekaran et al (2010);285:19637-19646; Wranik et al (2012) J Biol Chem 287:43331-43339; U.S.Pat. No. 5,932,448; WO 1993/011162; WO 2009/089004, and WO 2011/034605].As described herein, these methods may be used to generateALK7-Fc:ActRIIB-Fc heteromultimer complexes. See FIGS. 8-10 .

For example, one means by which interaction between specificpolypeptides may be promoted is by engineering protuberance-into-cavity(knob-into-holes) complementary regions such as described in Arathoon etal., U.S. Pat. No. 7,183,076 and Carter et al., U.S. Pat. No. 5,731,168.“Protuberances” are constructed by replacing small amino acid sidechains from the interface of the first polypeptide (e.g., a firstinteraction pair) with larger side chains (e.g., tyrosine ortryptophan). Complementary “cavities” of identical or similar size tothe protuberances are optionally created on the interface of the secondpolypeptide (e.g., a second interaction pair) by replacing large aminoacid side chains with smaller ones (e.g., alanine or threonine). Where asuitably positioned and dimensioned protuberance or cavity exists at theinterface of either the first or second polypeptide, it is onlynecessary to engineer a corresponding cavity or protuberance,respectively, at the adjacent interface.

At neutral pH (7.0), aspartic acid and glutamic acid are negativelycharged and lysine, arginine, and histidine are positively charged.These charged residues can be used to promote heterodimer formation andat the same time hinder homodimer formation. Attractive interactionstake place between opposite charges and repulsive interactions occurbetween like charges. In part, protein complexes disclosed herein makeuse of the attractive interactions for promoting heteromultimerformation (e.g., heterodimer formation), and optionally repulsiveinteractions for hindering homodimer formation (e.g., homodimerformation) by carrying out site directed mutagenesis of chargedinterface residues.

For example, the IgG1 CH3 domain interface comprises four unique chargeresidue pairs involved in domain-domain interactions: Asp356-Lys439′,Glu357-Lys370′, Lys392-Asp399′, and Asp399-Lys409′ [residue numbering inthe second chain is indicated by (′)]. It should be noted that thenumbering scheme used here to designate residues in the IgG1 CH3 domainconforms to the EU numbering scheme of Kabat. Due to the 2-fold symmetrypresent in the CH3-CH3 domain interactions, each unique interaction willrepresented twice in the structure (e.g., Asp-399-Lys409′ andLys409-Asp399′). In the wild-type sequence, K409-D399′ favors bothheterodimer and homodimer formation. A single mutation switching thecharge polarity (e.g., K409E; positive to negative charge) in the firstchain leads to unfavorable interactions for the formation of the firstchain homodimer. The unfavorable interactions arise due to the repulsiveinteractions occurring between the same charges (negative-negative;K409E-D399′ and D399-K409E′). A similar mutation switching the chargepolarity (D399K′; negative to positive) in the second chain leads tounfavorable interactions (K409′-D399K′ and D399K-K409′) for the secondchain homodimer formation. But, at the same time, these two mutations(K409E and D399K′) lead to favorable interactions (K409E-D399K′ andD399-K409′) for the heterodimer formation.

The electrostatic steering effect on heterodimer formation and homodimerdiscouragement can be further enhanced by mutation of additional chargeresidues which may or may not be paired with an oppositely chargedresidue in the second chain including, for example, Arg355 and Lys360.The table below lists possible charge change mutations that can be used,alone or in combination, to enhance ALK7:ActRIIB heteromultimerformation.

Examples of Pair-Wise Charged Residue Mutations to Enhance HeterodimerFormation Interacting Corresponding Position in Mutation in position inmutation in first chain first chain second chain second chain Lys409 Aspor Glu Asp399' Lys, Arg, or His Lys392 Asp or Glu Asp399' Lys, Arg, orHis Lys439 Asp or Glu Asp356' Lys, Arg, or His Lys370 Asp or Glu Glu357'Lys, Arg, or His Asp399 Lys, Arg, or His Lys409' Asp or Glu Asp399 Lys,Arg, or His Lys392' Asp or Glu Asp356 Lys, Arg, or His Lys439' Asp orGlu Glu357 Lys, Arg, or His Lys370' Asp or Glu

In some embodiments, one or more residues that make up the CH3-CH3interface in a fusion protein of the instant application are replacedwith a charged amino acid such that the interaction becomeselectrostatically unfavorable. For example, a positive-charged aminoacid in the interface (e.g., a lysine, arginine, or histidine) isreplaced with a negatively charged amino acid (e.g., aspartic acid orglutamic acid). Alternatively, or in combination with the forgoingsubstitution, a negative-charged amino acid in the interface is replacedwith a positive-charged amino acid. In certain embodiments, the aminoacid is replaced with a non-naturally occurring amino acid having thedesired charge characteristic. It should be noted that mutatingnegatively charged residues (Asp or Glu) to His will lead to increase inside chain volume, which may cause steric issues. Furthermore, Hisproton donor- and acceptor-form depends on the localized environment.These issues should be taken into consideration with the designstrategy. Because the interface residues are highly conserved in humanand mouse IgG subclasses, electrostatic steering effects disclosedherein can be applied to human and mouse IgG1, IgG2, IgG3, and IgG4.This strategy can also be extended to modifying uncharged residues tocharged residues at the CH3 domain interface.

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered to becomplementary on the basis of charge pairing (electrostatic steering).One of a pair of Fc sequences with electrostatic complementarity can bearbitrarily fused to the ALK7 or ActRIIB polypeptide of the construct,with or without an optional linker, to generate an ALK7:ActRIIBheteromultimer. This single chain can be coexpressed in a cell of choicealong with the Fc sequence complementary to the first Fc to favorgeneration of the desired multichain construct (e.g., ALK7:ActRIIBheteromultimer). In this example based on electrostatic steering, SEQ IDNO: 23 [human G1Fc(E134K/D177K)] and SEQ ID NO: 24 [humanG1Fc(K170D/K187D)] are examples of complementary Fc sequences in whichthe engineered amino acid substitutions are double underlined, and theTGF-beta superfamily type I or type II receptor polypeptide of theconstruct can be fused to either SEQ ID NO: 23 or SEQ ID NO: 24, but notboth. Given the high degree of amino acid sequence identity betweennative hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can beappreciated that amino acid substitutions at corresponding positions inhG2Fc, hG3Fc, or hG4Fc (see FIG. 5 ) will generate complementary Fcpairs which may be used instead of the complementary hG1Fc pair below(SEQ ID NOs: 23 and 24).

(SEQ ID NO: 23)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRKEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLKSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK (SEQ ID NO: 24)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYD TTPPVLDSDG SFFLYSDLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered forsteric complementarity. In part, the disclosure providesknobs-into-holes pairing as an example of steric complementarity. One ofa pair of Fc sequences with steric complementarity can be arbitrarilyfused to the ALK7 or ActRIIB polypeptide of the construct, with orwithout an optional linker, to generate an ALK7:ActRIIB heteromultimer.This single chain can be co-expressed in a cell of choice along with theFc sequence complementary to the first Fc to favor generation of thedesired multi-chain construct. In this example based on knobs-into-holespairing, SEQ ID NO: 25 [human G1Fc(T144Y)] and SEQ ID NO: 26 [humanG1Fc(Y185T)] are examples of complementary Fc sequences in which theengineered amino acid substitutions are double underlined, and the ALK7or ActRIIB polypeptide of the construct can be fused to either SEQ IDNO: 25 or SEQ ID NO: 26, but not both. Given the high degree of aminoacid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc,and native hG4Fc, it can be appreciated that amino acid substitutions atcorresponding positions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 5 ) willgenerate complementary Fc pairs which may be used instead of thecomplementary hG1Fc pair below (SEQ ID NOs: 25 and 26).

(SEQ ID NO: 25)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLYCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK (SEQ ID NO: 26)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLTSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK

An example of Fc complementarity based on knobs-into-holes pairingcombined with an engineered disulfide bond is disclosed in SEQ ID NO: 27[hG1Fc(S132C/T144W)] and SEQ ID NO: 28 [hG1Fc(Y127C/T144S/L146A/Y185V)].The engineered amino acid substitutions in these sequences are doubleunderlined, and the TGF-beta superfamily type I or type II polypeptideof the construct can be fused to either SEQ ID NO: 27 or SEQ ID NO: 28,but not both. Given the high degree of amino acid sequence identitybetween native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, itcan be appreciated that amino acid substitutions at correspondingpositions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 5 ) will generatecomplementary Fc pairs which may be used instead of the complementaryhG1Fc pair below (SEQ ID NOs: 27 and 28).

(SEQ ID NO: 27)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PCREEMTKNQ VSLWCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK (SEQ ID NO: 28)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVCTLP PSREEMTKNQ VSLSCAVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered togenerate interdigitating β-strand segments of human IgG and IgA C_(H)3domains. Such methods include the use of strand-exchange engineereddomain (SEED) C_(H)3 heterodimers allowing the formation of SEEDbodyfusion proteins [Davis et al. (2010) Protein Eng Design Sel 23:195-202].One of a pair of Fc sequences with SEEDbody complementarity can bearbitrarily fused to the ALK7 or ActIIB of the construct, with orwithout an optional linker, to generate an ALK7 or ActRIIB fusionpolypeptide. This single chain can be co-expressed in a cell of choicealong with the Fc sequence complementary to the first Fc to favorgeneration of the desired multi-chain construct. In this example basedon SEEDbody (Sb) pairing, SEQ ID NO: 29 [hG1Fc(Sb_(AG))] and SEQ ID NO:30 [hG1Fc(Sb_(GA))] are examples of complementary IgG Fc sequences inwhich the engineered amino acid substitutions from IgA Fc are doubleunderlined, and the ALK7 or ActRIIB polypeptide of the construct can befused to either SEQ ID NO: 29 or SEQ ID NO: 30, but not both. Given thehigh degree of amino acid sequence identity between native hG1Fc, nativehG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that aminoacid substitutions at corresponding positions in hG1Fc, hG2Fc, hG3Fc, orhG4Fc (see FIG. 5 ) will generate an Fc monomer which may be used in thecomplementary IgG-IgA pair below (SEQ ID NOs: 29 and 30).

(SEQ ID NO: 29)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PFRPEVHLLP PSREEMTKNQ VSLTCLARGF 151YPKDIAVEWE SNGQPENNYK TTPSRQEPSQ GTTTFAVTSK LTVDKSRWQQ 201GNVFSCSVMH EALHNHYTQK TISLSPGK (SEQ ID NO: 30)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PPSEELALNE LVTLTCLVKG 151FYPSDIAVEW ESNGQELPRE KYLTWAPVLD SDGSFFLYSI LRVAAEDWKK 201GDTFSCSVMH EALHNHYTQK SLDRSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains with a cleavable leucine zipper domainattached at the C-terminus of the Fc C_(H)3 domains. Attachment of aleucine zipper is sufficient to cause preferential assembly ofheterodimeric antibody heavy chains [Wranik et al (2012) J Biol Chem287:43331-43339]. As disclosed herein, one of a pair of Fc sequencesattached to a leucine zipper-forming strand can be arbitrarily fused tothe ALK7 or ActRIIB polypeptide of the construct, with or without anoptional linker, to generate an ALK7 or ActRIIB fusion polypeptide. Thissingle chain can be co-expressed in a cell of choice along with the Fcsequence attached to a complementary leucine zipper-forming strand tofavor generation of the desired multi-chain construct. Proteolyticdigestion of the construct with the bacterial endoproteinase Lys-C postpurification can release the leucine zipper domain, resulting in an Fcconstruct whose structure is identical to that of native Fc. In thisexample based on leucine zipper pairing, SEQ ID NO: 36 [hG1Fc-Ap1(acidic)] and SEQ ID NO: 37 [hG1Fc-Bp1 (basic)] are examples ofcomplementary IgG Fc sequences in which the engineered complimentaryleucine zipper sequences are underlined, and the ALK7 or ActRIIBpolypeptide of the construct can be fused to either SEQ ID NO: 36 or SEQID NO: 37, but not both. Given the high degree of amino acid sequenceidentity between native hG1Fc, native hG2Fc, native hG3Fc, and nativehG4Fc, it can be appreciated that leucine zipper-forming sequencesattached, with or without an optional linker, to hG1Fc, hG2Fc, hG3Fc, orhG4Fc (see FIG. 5 ) will generate an Fc monomer which may be used in thecomplementary leucine zipper-forming pair below (SEQ ID NOs: 36 and 37).

(SEQ ID NO: 36)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGKGGSAQ LEKELQALEK ENAQLEWELQ 251 ALEKELAQGA T(SEQ ID NO: 37)   1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE  51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGKGGSAQ LKKKLQALKK KNAQLKWKLQ 251 ALKKKLAQGA T

As described above, various methods are known in the art that increasedesired pairing of Fc-containing fusion polypeptide chains in a singlecell line to produce a preferred asymmetric fusion protein at acceptableyields [Klein et al (2012) mAbs 4:653-663; and Spiess et al (2015)Molecular Immunology 67(2A): 95-106]. In addition, ALK7:ActRIIBheteromultimers may be generated using a combination of heavy and lightchain fusion proteins comprising either an ALK7 or ActRIIB polypeptide.For example, in some embodiments, an ALK7 polypeptide may be fused, withor without a linker domain, to an immunoglobulin heavy chain (IgG1,IgG2, IgG3, IgG4, IgM, IgA1, or IgA2) that comprises at least a portionof the C_(H)1 domain. Similarly, an ActRIIB polypeptide may be fused,with or without a linker domain, to an immunoglobulin light chain (kappaor lambda) that comprises at least a portion of the light chain constantdomain (C_(L)). In alternative embodiments, an ActRIIB polypeptide maybe fused, with or without a linker domain, to an immunoglobulin heavychain (IgG1, IgG2, IgG3, IgG4, IgM, IgA1, or IgA2) that comprises atleast a portion of the C_(H)1 domain, and an ALK7 polypeptide may befused, with or without a linker domain, to an immunoglobulin light chain(kappa or lambda) that comprises at least a portion of the light chainconstant domain (C_(L)). This design takes advantage of the naturalability of the heavy chains to heterodimerize with light chains. Inparticular, heterodimerization of a heavy and light chain occurs betweenthe C_(H)1 with the C_(L), which is generally stabilized by covalentlinking of the two domains via a disulfide bridge. Constructs employingthe full-length heavy chain, or at least a portion of the heavy chaincomprising the hinge region, could give rise to antibody-like moleculescomprising two “light chains” and two “heavy chains”. See FIG. 9 . Apotential advantage of this design is that it may more closely mimic thenaturally occurring ALK7-ligand-ActRIIB complex and may display higheraffinity for the ligand than comparable single heterodimers. In someembodiments, this design may be modified by incorporating various heavychain truncations including, for example, truncations that comprise theC_(H)1 domain and some or all of the hinge domain (giving rise toF(ab′)₂-like molecules) as well as truncations that only comprise theC_(H)1 domain or a fragment thereof (giving rise to Fab-like molecules).See FIG. 9G. Various methods for designing such heteromultimerconstructs are described in US 2009/0010879, Klein et al [(2012) mAbs4:653-663], and Spiess et al [(2015) Molecular Immunology 67(2A):95-106] the contents of which are incorporated in their entirety herein.

In some embodiments, it is desirable to generate antibody-likeALK7:ActRIIB heterodimers comprising at least one branch of the complexcomprising an ALK7-C_(L):ActRIIB-C_(H)1 heterodimer pair and at least asecond branch comprising an ActRIIB-C_(L):ALK7-C_(H)1 heterodimer pair.See, e.g., FIG. 9B. Such heterodimer complexes can be generated, forexample, using combinations of heavy chain and light chain asymmetricalpairing technologies [Spiess et al (2015) Molecular Immunology 67(2A):95-106]. For example, in CrossMab technology, [Schaefer et al (2011).Proc. Natl. Acad. Sci. U.S.A. 108: 11187-11192] light chain mispairingis overcome using domain crossovers and heavy chains heterodimerizedusing knobs-into-holes [Merchant et al (1998) Nat. Biotechnol. 16:677-681]. For the domain crossovers either the variable domains or theconstant domains are swapped between light and heavy chains to createtwo asymmetric Fab arms that drive cognate light chain pairing whilepreserving the structural and functional integrity of the variabledomain [Fenn et al (2013) PLoS ONE 8: e61953]. An alternative approachfor overcoming light chain mispairing is designing heavy and lightchains with orthogonal Fab inter-faces [Lewis (2014) Nat. Biotechnol.32: 191-198]. This has been accomplished by computational modeling [Daset al (2008) Annu. Rev. Biochem. 77: 363-382] in combination with X-raycrystallography to identify mutations at the V_(H)/V_(L) andC_(H)1/C_(L) interfaces. For the heterodimers generated using thismethodology, it may be necessary to engineer mutations into bothV_(H)/V_(L) and C_(H)1/C_(L) interfaces to minimize heavy/light chainmispairing. The designed orthogonal Fab interface may be used inconjunction with a heavy chain heterodimerization strategy to facilitateefficient IgG production in a single host cell. Electrostatic steeringmay also be used to generate orthogonal Fab interfaces to facilitate theconstruction of such heterodimers. Peptide linkers may be used to ensurecognate pairing of light and heavy chains in a format known as “LUZ-Y”[Wranik et al (2012) J. Biol. Chem. 287: 43331-43339], wherein heavychain heterodimerization is accomplished using leucine zippers which maybe subsequently removed by proteolysis in vitro.

Alternatively, ALK7:ActRIIB heteromultimers may comprise one or moresingle-chain ligand traps as described herein, optionally which may becovalently or non-covalently associated with one or more ALK7 or ActRIIBpolypeptides as well as additional ALK7:ActRIIB single chain ligandtraps [US 2011/0236309 and US2009/0010879]. See FIG. 12 . As describedherein, single-chain ligand traps do not require fusion to anymultimerization domain such as coiled-coil Fc domains to be multivalent.In general, single-chain ligand traps of the present disclosure compriseat least one ALK7 polypeptide domain and one ActRIIB polypeptide domain.The ALK7 and ActRIIB polypeptide domains, generally referred to hereinas binding domains (BD), optionally may be joined by a linker region.

For example, in one aspect, the present disclosure providesheteromultimers comprising a polypeptide having the following structure:

(<BD1>-linker1)_(k)-[<BD2>-linker2-{<BD3>-linker3}_(f)]_(n)-(<BD4>)_(m)-(linker4-BD5>_(d))_(h)

where: n and h are independently greater than or equal to one; d, f, m,and k are independently equal to or greater than zero; BD1, BD2, BD3,BD4, and BD5 are independently ALK7 or ActRIIB polypeptide domains,wherein at least one of BD1, BD2, BD3, and BD4 is an ALK7 polypeptidedomain, and wherein at least one of BD1, BD2, BD3, and BD4 is an ActRIIBpolypeptide domain, and linker1, linker2, linker3, and linker 4 areindependently greater than or equal to zero. In some embodiment,ALK7:ActRIIB single-chain traps comprise at least two different ALK7polypeptides. In some embodiments, ALK7:ActRIIB single-chain trapscomprise at least two different ActRIIB polypeptides. In someembodiment, ALK7:ActRIIB single-chain traps comprise at least twodifferent linkers. Depending on the values of selected for d, f, h, k,m, and n, the heteromultimer structure may comprise a large number ofrepeating units in various combinations or may be a relatively simplestructure.

In another aspect, the present disclosure provides heteromultimerscomprising a polypeptide having the following structure:

<BD1>-linker1-<BD2>

In yet another aspect, the present disclosure provides heteromultimerscomprising a polypeptide having the following structure:

<BD1>-(linker2-<BD2>)_(n)

where n is greater than or equal one.

Another aspect of the invention provides heteromultimers comprising apolypeptide having the following structure:

(<BD1>-linker1-<BD1>)_(f)-linker2-(<BD2>-linker3-<BD3>)_(g)

wherein f and g are greater than or equal to one.

In an embodiment where BD2 and BD3 are the same, and f and g are thesame number, this can result in a substantially mirror symmetricstructure around linker 2, subject to differences in the linkers. Ininstances where BD2 is different from BD3 and/or where f and g aredifferent numbers, different structures will be produced. It is withinthe capacity of one of ordinary skill in the art to select suitablebinding domains, linkers, and repeat frequencies in light of thedisclosure herein and knowledge in the art. Specific, non-limitingexamples of such single-chain ligand traps in accordance with thepresent disclosure are represented schematically in FIG. 11 .

The linkers (1, 2, 3, and 4) may be the same or different. The linkerregion provides a segment that is distinct from the structuredligand-binding domains of ALK7 and ActRIIB and thus can be used forconjugation to accessory molecules (e.g., molecules useful in increasingstability such as PEGylation moieties) without having to chemicallymodify the binding domains. The linker may include an unstructured aminoacid sequence that may be either the same as or derived fromconservative modifications to the sequence of a natural unstructuredregion in the extracellular portion of the receptor for the ligand ofinterest or another receptor in the TGF-β superfamily. In otherinstances, such linkers may be entirely artificial in composition andorigin but will contain amino acids selected to provide an unstructuredflexible linker with a low likelihood of encountering electrostatic orsteric hindrance complications when brought into close proximity to theligand of interest. Linker length will be considered acceptable when itpermits binding domains located on each of the N- and C-termini of thelinker to bind their natural binding sites on their natural ligand suchthat, with both binding domains so bound, the ligand is bound with ahigher affinity than it would be bound by binding of only one of thebinding domains. In some instances, the number of amino acid residues inthe linker of either natural or artificial origin is selected to beequal to or greater than the minimum required distance for simultaneous(bridged) binding to two binding sites on the ALK7 and/or ActRIIBligand. For example, and without wishing to be limiting in any manner,the linker length may be between about 1-10 amino acids, 10-20 aminoacids, 18-80 amino acids, 25-60 amino acids, 35-45 amino acids, or anyother suitable length.

Linkers may be designed to facilitate purification of the polypeptide.The exact purification scheme chosen will determine what modificationsare needed, for example and without wishing to be limiting, additions ofpurification “tags” such as His tags is contemplated; in other examples,the linker may include regions to facilitate the addition of cargo oraccessory molecules. When such additions affect the unstructured natureof the linker or introduce potential electrostatic or steric concerns,appropriate increases to the linker length will be made to ensure thatthe two binding domains are able to bind their respective sites on theligand. In light of the methods and teachings herein, suchdeterminations could be made routinely by one skilled in the art.

In addition, the present design permits linkage of other cargo molecules(for example imaging agents like fluorescent molecules), toxins, etc.For example, and without wishing to be limiting in any manner,single-chain polypeptides can be modified to add one or more cargoand/or accessory molecules (referred to collectively herein by R1, R2,R3, R4, etc.):

Without limiting the generality of R substituents available, R1, R2, R3,R4, R5, R6, R7, R8, R9, may or may not be present; when present, theymay be the same or different, and may independently be one or more of: afusion protein for targeting, for example, but not limited to such as anantibody fragment (e.g. single chain Fv) and/or a single domain antibody(sdAb); a radiotherapy and/or imaging agent, for example, but notlimited to a radionucleotide (e.g. ¹²³I, ¹¹¹In, ¹⁸F, ⁶⁴C, ⁶⁸Y, ¹²⁴I,¹³¹I, ⁹⁰Y, ¹⁷⁷Lu, ⁵⁷Cu, ²¹³Bi, ²¹¹At), a fluorescent dye (e.g. AlexaFluor, Cy dye) and/or a fluorescent protein tag (e.g. GFP, DsRed); acytotoxic agent for chemotherapy, for example, but not limited todoxorubicin, calicheamicin, a maytansinoid derivatives (e.g. DM1, DM4),a toxin (eg. truncated Pseudomonas endotoxin A, diptheria toxin); ananoparticle-based carrier, for example, but not limited to polyethyleneglycol (PEG), a polymer-conjugated to drug, nanocarrier or imaging agent(e.g. of a polymer N-(2-hydroxylpropyl) methacrylamide (HPMA), glutamicacid, PEG, dextran); a drug (for example, but not limited todoxorubicin, camptothecin, paclitaxel, palatinate); a nanocarrier, forexample, but not limited to a nanoshell or liposome; an imaging agent,for example, but not limited to Supermagnetic Iron Oxide (SPIO); adendrimer; and/or a solid support for use in ligand purification,concentration or sequestration (e.g. nanoparticles, inert resins,suitable silica supports).

In general, it will not be preferable to have cargo or accessorymolecules in all possible positions, as this may cause steric orelectrostatic complications. However, the effects of adding a cargo oraccessory molecule to any given position or positions on the structurecan be determined routinely in light of the disclosure herein bymodeling the linker between the binding domains and carrying outmolecular dynamics simulations to substantially minimize molecularmechanics energy and reduce steric and electrostatic incompatibilitybetween the linker and the ALK7 and ActRIIB polypeptides as taughtherein.

It may be preferable to add the cargo or accessory molecule to thelinker portion of the agent, rather to the binding domain, to reduce thelikelihood of interference in binding function. However, addition to thebinding domain is possible and could be desirable in some instances andthe effect of such an addition can be determined routinely in advance bymodeling the binding agent and the linker with the proposed addition asdescribed herein.

Conjugation methodologies may be performed using commercial kits thatenable conjugation via common reactive groups such as primary amines,succinimidyl (NHS) esters and sulfhydryl-reactive groups. Somenon-limiting examples are: Alexa Fluor 488 protein labeling kit(Molecular Probes, Invitrogen detection technologies) and PEGylationkits (Pierce Biotechnology Inc.).

In certain aspects, ALK7:ActRIIB single-chain traps may be covalently ornon-covalently associated with one or more ALK7 or ActRIIB polypeptidesas well as additional ALK7:ActRIIB single chain ligand traps to formhigher order heteromultimers, which may be used in accordance with themethods described herein. See, e.g., FIG. 12 . For example, anALK7:ActRIIB single chain ligand trap may further comprise amultimerization domain as described herein. In some embodiments,ALK7:ActRIIB single chain ligand traps comprise a constant domain of anIg immunoglobulin. Such immunoglobulins constant domains may be selectedto promote symmetrical or asymmetrical complexes comprising at least onesingle-chain ALK7:ActRIIB trap.

In certain aspects, an ALK7:ActRIIB single-chain trap, or combinationsof such traps, may be used as ALK7:ActRIIB antagonists to treat orprevent an ALK7:ActRIIB disorder or disease as described herein (e.g.,kidney disease and/or a metabolic disorder or condition).

It is understood that different elements of the fusion proteins (e.g.,immunoglobulin Fc fusion proteins) may be arranged in any manner that isconsistent with desired functionality. For example, an ALK7 and/orActRIIB polypeptide domain may be placed C-terminal to a heterologousdomain, or alternatively, a heterologous domain may be placed C-terminalto an ALK7 and/or ActRIIB polypeptide domain. The ALK7 and/or ActRIIBpolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

For example, an ALK7 and/or ActRIIB receptor fusion protein may comprisean amino acid sequence as set forth in the formula A-B-C. The B portioncorresponds to an ALK7 or ActRIIB polypeptide domain. The A and Cportions may be independently zero, one, or more than one amino acid,and both the A and C portions when present are heterologous to B. The Aand/or C portions may be attached to the B portion via a linkersequence. A linker may be rich in glycine (e.g., 2-10, 2-5, 2-4, 2-3glycine residues) or glycine and proline residues and may, for example,contain a single sequence of threonine/serine and glycines or repeatingsequences of threonine/serine and/or glycines, e.g., GGG (SEQ ID NO:13), GGGG (SEQ ID NO: 14), TGGGG (SEQ ID NO: 15), SGGGG (SEQ ID NO: 16),TGGG (SEQ ID NO: 17), SGGG (SEQ ID NO: 18), or GGGGS (SEQ ID NO: 58)singlets, or repeats. In certain embodiments, an ALK7 and/or ActRIIBfusion protein comprises an amino acid sequence as set forth in theformula A-B-C, wherein A is a leader (signal) sequence, B consists of anALK7 and/or ActRIIB polypeptide domain, and C is a polypeptide portionthat enhances one or more of in vivo stability, in vivo half-life,uptake/administration, tissue localization or distribution, formation ofprotein complexes, and/or purification. In certain embodiments, an ALK7and/or ActRIIB fusion protein comprises an amino acid sequence as setforth in the formula A-B-C, wherein A is a TPA leader sequence, B is anALK7 or ActRIIB receptor polypeptide domain, and C is an immunoglobulinFc domain. Preferred fusion proteins comprise the amino acid sequenceset forth in any one of SEQ ID NOs: 71, 73, 74, 76, 77, 78, 79 and 80.

In some embodiments, ALK7:ActRIIB heteromultimers further comprise oneor more heterologous portions (domains) so as to confer a desiredproperty. For example, some fusion domains are particularly useful forisolation of the fusion proteins by affinity chromatography. Well-knownexamples of such fusion domains include, but are not limited to,polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin,protein A, protein G, an immunoglobulin heavy-chain constant region(Fc), maltose binding protein (MBP), or human serum albumin. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Many of such matrices are availablein “kit” form, such as the Pharmacia GST purification system and theQIAexpress™ system (Qiagen) useful with (HIS₆) fusion partners (“HIS₆”disclosed as SEQ ID NO: 100). As another example, a fusion domain may beselected so as to facilitate detection of the ligand trap polypeptides.Examples of such detection domains include the various fluorescentproteins (e.g., GFP) as well as “epitope tags,” which are usually shortpeptide sequences for which a specific antibody is available. Well-knownepitope tags for which specific monoclonal antibodies are readilyavailable include FLAG, influenza virus haemagluttinin (HA), and c-myctags. In some cases, the fusion domains have a protease cleavage site,such as for factor Xa or thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation.

In certain embodiments, ALK7 and/or ActRIIB polypeptides may contain oneor more modifications that are capable of stabilizing the polypeptides.For example, such modifications enhance the in vitro half-life of thepolypeptides, enhance circulatory half-life of the polypeptides, and/orreduce proteolytic degradation of the polypeptides. Such stabilizingmodifications include, but are not limited to, fusion proteins(including, for example, fusion proteins comprising an ALK7 and/orActRIIB polypeptide domain and a stabilizer domain), modifications of aglycosylation site (including, for example, addition of a glycosylationsite to a polypeptide of the disclosure), and modifications ofcarbohydrate moiety (including, for example, removal of carbohydratemoieties from a polypeptide of the disclosure). As used herein, the term“stabilizer domain” not only refers to a fusion domain (e.g., animmunoglobulin Fc domain) as in the case of fusion proteins, but alsoincludes nonproteinaceous modifications such as a carbohydrate moiety,or nonproteinaceous moiety, such as polyethylene glycol.

In preferred embodiments, ALK7:ActRIIB heteromultimers to be used inaccordance with the methods described herein are isolated complexes. Asused herein, an isolated protein (or protein complex) or polypeptide (orpolypeptide complex) is one which has been separated from a component ofits natural environment. In some embodiments, a heteromultimer of thedisclosure is purified to greater than 95%, 96%, 97%, 98%, or 99% purityas determined by, for example, electrophoretic (e.g., SDS-PAGE,isoelectric focusing (IEF), capillary electrophoresis) orchromatographic (e.g., ion exchange or reverse phase HPLC). Methods forassessment of antibody purity are well known in the art [Flatman et al.,(2007) J. Chromatogr. B 848:79-87]. In some embodiments, ALK7:ActRIIBheteromultimer preparations are substantially free of ALK7 and/orActRIIB homomultimers. For example, in some embodiments, ALK7:ActRIIBheteromultimers preparations comprise less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than about 1% ALK7 homomultimers. In someembodiments, ALK7:ActRIIB heteromultimer preparations comprise less thanabout 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% ActRIIBhomomultimers. In some embodiments, ALK7:ActRIIB heteromultimerpreparations comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or less than about 1% ALK7 homomultimers and less than about 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% ActRIIBhomomultimers.

In certain embodiments, ALK7 and/or ActRIIB polypeptides, as well asheteromultimers comprising the same, can be produced by a variety ofart-known techniques. For example, polypeptides can be synthesized usingstandard protein chemistry techniques such as those described inBodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin(1993) and Grant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H.Freeman and Company, New York (1992). In addition, automated peptidesynthesizers are commercially available (Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, the polypeptides and complexes,including fragments or variants thereof, may be recombinantly producedusing various expression systems [E. coli, Chinese Hamster Ovary (CHO)cells, COS cells, baculovirus] as is well known in the art. In a furtherembodiment, the modified or unmodified polypeptides may be produced bydigestion of recombinantly produced full-length ALK7 and/or ActRIIBpolypeptides by using, for example, a protease, e.g., trypsin,thermolysin, chymotrypsin, pepsin, or paired basic amino acid convertingenzyme (PACE). Computer analysis (using commercially available software,e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be usedto identify proteolytic cleavage sites.

B. Nucleic Acids Encoding ALK7 and/or ActRIIB Polypeptides

In certain embodiments, the present disclosure provides isolated and/orrecombinant nucleic acids encoding ALK7 and/or ActRIIB receptors(including fragments, functional variants, and fusion proteins thereof)disclosed herein. For example, SEQ ID NO: 11 encodes a naturallyoccurring human ALK7 precursor polypeptide, while SEQ ID NO: 12 encodesa mature extracellular domain of ALK7. The subject nucleic acids may besingle-stranded or double stranded. Such nucleic acids may be DNA or RNAmolecules. These nucleic acids may be used, for example, in methods formaking ALK7:ActRIIB heteromultimers as described herein.

As used herein, isolated nucleic acid(s) refers to a nucleic acidmolecule that has been separated from a component of its naturalenvironment. An isolated nucleic acid includes a nucleic acid moleculecontained in cells that ordinarily contain the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

In certain embodiments, nucleic acids encoding ALK7 and/or ActRIIBpolypeptides of the present disclosure are understood to include any oneof SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 41, 44, 45, 72, or 75, as wellas variants thereof. Variant nucleotide sequences include sequences thatdiffer by one or more nucleotide substitutions, additions, or deletionsincluding allelic variants, and therefore, will include coding sequencesthat differ from the nucleotide sequence designated in any one of SEQ IDNOs: 7, 8, 11, 12, 21, 22, 40, 41, 44, 45, 72, or 75.

In certain embodiments, TGFβ superfamily ALK7 and/or ActRIIBpolypeptides of the present disclosure are encoded by isolated orrecombinant nucleic acid sequences that comprise, consist essentiallyof, or consists of a sequence that is least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 41, 44, 45, 72, or75. One of ordinary skill in the art will appreciate that nucleic acidsequences that comprise, consist essentially of, or consists of asequence complementary to a sequence that is least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 41, 44, 45, 72,or 75 also within the scope of the present disclosure. In furtherembodiments, the nucleic acid sequences of the disclosure can beisolated, recombinant, and/or fused with a heterologous nucleotidesequence or in a DNA library.

In other embodiments, nucleic acids of the present disclosure alsoinclude nucleotide sequences that hybridize under stringent conditionsto the nucleotide sequence designated in SEQ ID NOs: 7, 8, 11, 12, 21,22, 40, 41, 44, 45, 72, or 75, the complement sequence of SEQ ID NOs: 7,8, 11, 12, 21, 22, 40, 41, 44, 45, 72, or 75, or fragments thereof. Oneof ordinary skill in the art will understand readily that appropriatestringency conditions which promote DNA hybridization can be varied. Forexample, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the disclosure provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 41, 44, 45, 72, or 75 todegeneracy in the genetic code are also within the scope of thedisclosure. For example, a number of amino acids are designated by morethan one triplet. Codons that specify the same amino acid, or synonyms(for example, CAU and CAC are synonyms for histidine) may result in“silent” mutations which do not affect the amino acid sequence of theprotein. However, it is expected that DNA sequence polymorphisms that dolead to changes in the amino acid sequences of the subject proteins willexist among mammalian cells. One skilled in the art will appreciate thatthese variations in one or more nucleotides (up to about 3-5% of thenucleotides) of the nucleic acids encoding a particular protein mayexist among individuals of a given species due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of this disclosure.

In certain embodiments, the recombinant nucleic acids of the disclosuremay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the disclosure. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In some embodiments, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selectable marker genes are well known in the art and will vary with thehost cell used.

In certain aspects, the subject nucleic acid is provided in anexpression vector comprising a nucleotide sequence encoding an ALK7and/or ActRIIB polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of ALK7 and/or ActRIIB polypeptide.Accordingly, the term regulatory sequence includes promoters, enhancers,and other expression control elements. Exemplary regulatory sequencesare described in Goeddel; Gene Expression Technology: Methods inEnzymology, Academic Press, San Diego, CA (1990). For instance, any of awide variety of expression control sequences that control the expressionof a DNA sequence when operatively linked to it may be used in thesevectors to express DNA sequences encoding a ALK7 and/or ActRIIBpolypeptides. Such useful expression control sequences, include, forexample, the early and late promoters of SV40, tet promoter, adenovirusor cytomegalovirus immediate early promoter, RSV promoters, the lacsystem, the trp system, the TAC or TRC system, T7 promoter whoseexpression is directed by T7 RNA polymerase, the major operator andpromoter regions of phage lambda, the control regions for fd coatprotein, the promoter for 3-phosphoglycerate kinase or other glycolyticenzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters ofthe yeast a-mating factors, the polyhedron promoter of the baculovirussystem and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof. It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other protein encoded by thevector, such as antibiotic markers, should also be considered.

A recombinant nucleic acid of the present disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant ALK7 and/or ActRIIB polypeptides include plasmids andother vectors. For instance, suitable vectors include plasmids of thefollowing types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures [Molecular Cloning A Laboratory Manual, 3rd Ed.,ed. by Sambrook, Fritsch and Maniatis Cold Spring Harbor LaboratoryPress, 2001]. In some instances, it may be desirable to express therecombinant polypeptides by the use of a baculovirus expression system.Examples of such baculovirus expression systems include pVL-derivedvectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors(such as pAcUW1), and pBlueBac-derived vectors (such as the B-galcontaining pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ALK7 and/or ActRIIB polypeptides in CHO cells, such as aPcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors(Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison,Wisc.). As will be apparent, the subject gene constructs can be used tocause expression of the subject ALK7 and/or ActRIIB polypeptide in cellspropagated in culture, e.g., to produce proteins, including fusionproteins or variant proteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence for one or more of thesubject ALK7 and/or ActRIIB polypeptides. The host cell may be anyprokaryotic or eukaryotic cell. For example, an ALK7 and/or ActRIIBpolypeptide may be expressed in bacterial cells such as E. coli, insectcells (e.g., using a baculovirus expression system), yeast, or mammaliancells [e.g. a Chinese hamster ovary (CHO) cell line]. Other suitablehost cells are known to those skilled in the art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject ALK7 and/or ActRIIB polypeptides. For example, ahost cell transfected with an expression vector encoding an ALK7 and/orActRIIB polypeptide can be cultured under appropriate conditions toallow expression of the ALK7 and/or ActRIIB polypeptide to occur. Thepolypeptide may be secreted and isolated from a mixture of cells andmedium containing the polypeptide. Alternatively, ALK7 and/or ActRIIBpolypeptide may be isolated from a cytoplasmic or membrane fractionobtained from harvested and lysed cells. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. The subject polypeptides can be isolated fromcell culture medium, host cells, or both, using techniques known in theart for purifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis,immunoaffinity purification with antibodies specific for particularepitopes of ALK7 and/or ActRIIB polypeptides and affinity purificationwith an agent that binds to a domain fused to ALK7 and/or ActRIIBpolypeptide (e.g., a protein A column may be used to purify ALK7-Fcand/or ActRIIB-Fc fusion proteins). In some embodiments, the ALK7 and/orActRIIB polypeptide is a fusion protein containing a domain whichfacilitates its purification.

In some embodiments, purification is achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. An ALK7and/or ActRIIB polypeptide, as well as fusion proteins and heteromericcomplexes thereof, may be purified to a purityof >90%, >95%, >96%, >98%, or >99% as determined by size exclusionchromatography and >90%, >95%, >96%, >98%, or >99% as determined by SDSPAGE. The target level of purity should be one that is sufficient toachieve desirable results in mammalian systems, particularly non-humanprimates, rodents (mice), and humans.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ALK7 and/orActRIIB polypeptide, can allow purification of the expressed fusionprotein by affinity chromatography using a Ni²⁺ metal resin. Thepurification leader sequence can then be subsequently removed bytreatment with enterokinase to provide the purified ALK7 and/or ActRIIBpolypeptide, as well as heteromultimers thereof [Hochuli et al. (1987)J. Chromatography 411:177; and Janknecht et al. (1991) PNAS USA88:8972].

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence. See,e.g., Current Protocols in Molecular Biology, eds. Ausubel et al., JohnWiley & Sons: 1992.

C. Antibody Antagonists

In certain aspects, an ALK7:ActRIIB antagonist is an antibody(ALK7:ActRIIB antagonist antibody), or combination of antibodies. AnALK7:ActRIIB antagonist antibody, or combination of antibodies, may bindto, for example, one or more ALK7 ligands, ActRIIB ligands,ALK7:ActRIIB-binding ligands, an ALK7 receptor, an ActRIIB receptor,and/or a TGF-beta superfamily co-receptor (e.g., Crypto or Cryptic). Asdescribed herein, ALK7:ActRIIB antagonist antibodies may be used, aloneor in combination with one or more supportive therapies or activeagents, to treat a patient in need thereof (e.g., patients having kidneydisease and/or a metabolic disorder or condition).

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits one or more of the ligandsbound or likely bound by an ALK7:ActRIIB heteromultimer, such as activinB, GDF11, activin A, BMP10, BMP6, BMP5, GDF3, activin C, activin E,activin AC, activin BC, activin AE, nodal, or activin BE. Therefore, insome embodiments, an ALK7:ActRIIB antagonist antibody, or combination ofantibodies, binds to at least one of such ligands. As an example, asused herein, an activin C antibody (or anti-activin C antibody)generally refers to an antibody that can bind to activin C withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting activin C. In certain embodiments,the extent of binding of a activin C antibody to an unrelated,non-activin C protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, or less than about 1% of the binding of the antibody to activinC as measured, for example, by a radioimmunoassay (MA), Biacore, orother protein interaction or binding affinity assay. In certainembodiments, an activin C antibody binds to an epitope of activin C thatis conserved among activin C from different species. In certainpreferred embodiments, an anti-activin C antibody binds to human activinC. In some embodiments, an activin C antibody may inhibit activin C frombinding to a type I and/or type II receptor (e.g., ActRIIB and/or ALK7)and thus inhibit activin C-mediated signaling (e.g., Smad signaling). Insome embodiments, an activin C antibody may inhibit activin C frombinding to a co-receptor and thus inhibit activin C-mediated signaling(e.g., Smad signaling). It should be noted that activin C shares somesequence homology to activin A, B and E and therefore antibodies thatbind to activin C, in some instances, may also bind to and/or inhibitanother activin. In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to, for example, activin C and further binds to, for example,one or more additional TGF-β superfamily ligands that bind toALK7:ActRIIB heteromultimer [e.g., activin (e.g., activin A, activin B,activin C, activin E, activin AB, activin AC, activin BC, activin AE, oractivin BE), GDF11, GDF8, BMP10, BMP6, BMP5, nodal, and GDF3], one ormore type I receptor and/or type II receptors (e.g., ActRIIB and/orALK7), and/or one or more co-receptors. In some embodiments, amultispecific antibody that binds to activin C does not bind or does notsubstantially bind to BMP9 (e.g., binds to BMP9 with a K_(D) of greaterthan 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M orabout 1×10⁻⁹ M). In some embodiments, the disclosure relates tocombinations of antibodies, and uses thereof, wherein the combination ofantibodies comprises a combination of antibodies that bind to, forexample, two or more TGF-β superfamily ligand that bind to ALK7:ActRIIBheteromultimer [e.g., activin (e.g., activin A, activin B, activin C,activin E, activin AB, activin AC, activin BC, activin AE, or activinBE), GDF11, GDF8, BMP10, BMP6, BMP5, nodal, and GDF] one or more type Ireceptor and/or type II receptors (e.g., ActRIIB and/or ALK7), and/orone or more co-receptors. In some embodiments, a combination ofantibodies does not comprise a BMP9 antibody.

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least GDF8. Therefore, insome embodiments, an ALK7:ActRIIB antagonist antibody, or combination ofantibodies, binds to at least GDF8. As used herein, a GDF8 antibody (oranti-GDF8 antibody) generally refers to an antibody that binds to GDF8with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting GDF8. In certainembodiments, the extent of binding of a GDF8 antibody to an unrelated,non-GDF8 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or less than about 1% of the binding of the antibody to GDF8 asmeasured, for example, by a radioimmunoassay (MA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, aGDF8 antibody binds to an epitope of GDF8 that is conserved among GDF8from different species. In certain preferred embodiments, an anti-GDF8antibody binds to human GDF8. In some embodiments, a GDF8 antibody mayinhibit GDF8 from binding to a type I and/or type II receptor (e.g.,ActRIIB and/or ALK7) and thus inhibit GDF8-mediated signaling (e.g.,Smad signaling). In some embodiments, a GDF8 antibody may inhibit GDF8from binding to a co-receptor and thus inhibit GDF8-mediated signaling(e.g., Smad signaling). It should be noted that GDF8 has high sequencehomology to GDF11 and therefore antibodies that bind to GDF8, in someinstances, may also bind to and/or inhibit GDF11. In some embodiments,the disclosure relates to a multispecific antibody (e.g., bi-specificantibody), and uses thereof, that binds to GDF8 and further binds to,for example, one or more additional TGF-β superfamily ligands [e.g.,activin (e.g., activin A, activin B, activin C, activin E, activin AB,activin AC, activin BC, activin AE, activin BE), GDF11, BMP10, BMP6,BMP5, nodal, and GDF3], one or more type I receptor and/or type IIreceptors (e.g., ActRIIB and/or ALK7), and/or one or more co-receptors.In some embodiments, a multispecific antibody that binds to GDF8 doesnot bind or does not substantially bind to BMP9 (e.g., binds to BMP9with a K_(D) of greater than 1×10⁻⁷M or has relatively modest binding,e.g., about 1×10⁻⁸M or about 1×10⁻⁹M). In some embodiments, thedisclosure relates to combinations of antibodies, and uses thereof,wherein the combination of antibodies comprises a GDF8 antibody and oneor more additional antibodies that bind to, for example, one or moreadditional TGF-β superfamily ligand [e.g., activin (e.g., activin A,activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE, activin BE), GDF11, GDF3, BMP6, BMP10, nodal, and BMP5], oneor more type I receptor and/or type II receptors (e.g., ActRIIB and/orALK7), and/or one or more co-receptors. In some embodiments, acombination of antibodies that comprises a GDF8 antibody does notcomprise a BMP9 antibody.

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least GDF11. Therefore,in some embodiments, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, binds to at least GDF11. As used herein, a GDF11 antibody(or anti-GDF11 antibody) generally refers to an antibody that binds toGDF11 with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting GDF11. In certainembodiments, the extent of binding of a GDF11 antibody to an unrelated,non-GDF11 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or less than about 1% of the binding of the antibody to GDF11 asmeasured, for example, by a radioimmunoassay (MA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, aGDF11 antibody binds to an epitope of GDF11 that is conserved amongGDF11 from different species. In certain preferred embodiments, ananti-GDF11 antibody binds to human GDF11. In some embodiments, a GDF11antibody may inhibit GDF11 from binding to a type I and/or type IIreceptor (e.g., ActRIIB and/or ALK7) and thus inhibit GDF11-mediatedsignaling (e.g., Smad signaling). In some embodiments, a GDF11 antibodymay inhibit GDF11 from binding to a co-receptor and thus inhibitGDF11-mediated signaling (e.g., Smad signaling). It should be noted thatGDF11 has high sequence homology to GDF8 and therefore antibodies thatbind to GDF11, in some instances, may also bind to and/or inhibit GDF8.In some embodiments, the disclosure relates to a multispecific antibody(e.g., bi-specific antibody), and uses thereof, that binds to GDF11 andfurther binds to, for example, one or more additional TGF-superfamilyligands [e.g., activin (e.g., activin A, activin B, activin C, activinE, activin AB, activin AC, activin BC, activin AE, activin BE), GDF11,BMP10, BMP6, BMP5, nodal, and GDF3], one or more type I receptor and/ortype II receptors (e.g., ActRIIB and/or ALK7), and/or one or moreco-receptors. In some embodiments, a multispecific antibody that bindsto GDF11 does not bind or does not substantially bind to BMP9 (e.g.,binds to BMP9 with a K_(D) of greater than 1×10⁻⁷M or has relativelymodest binding, e.g., about 1×10⁻⁸M or about 1×10⁻⁹M). In someembodiments, the disclosure relates to combinations of antibodies, anduses thereof, wherein the combination of antibodies comprises a GDF11antibody and one or more additional antibodies that bind to, forexample, one or more additional TGF-β superfamily ligand [e.g., activin(e.g., activin A, activin B, activin C, activin E, activin AB, activinAC, activin BC, activin AE, activin BE), GDF8, GDF3, BMP6, BMP10, nodal,and BMP5], one or more type I receptor and/or type II receptors (e.g.,ActRIIB and/or ALK7), and/or one or more co-receptors. In someembodiments, a combination of antibodies that comprises a GDF11 antibodydoes not comprise a BMP9 antibody.

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least activin (activin A,activin B, activin C, activin E, activin AB, activin AC, activin AE,activin BC and/or activin BE). Therefore, in some embodiments, anALK7:ActRIIB antagonist antibody, or combination of antibodies, binds toat least activin (activin A, activin B, activin C, activin E, activinAB, activin AC, activin AE, activin BC and/or activin BE). As usedherein, an activin antibody (or anti-activin antibody) generally refersto an antibody that can bind to a form of activin with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting that form of activin. In certainembodiments, the extent of binding of an activin antibody to anunrelated, non-activin protein is less than about 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody toactivin as measured, for example, by a radioimmunoassay (MA), Biacore,or other protein interaction or binding affinity assay. In certainembodiments, an activin antibody binds to an epitope of activin that isconserved among activin from different species. In certain preferredembodiments, an anti-activin antibody binds to human activin. In otherpreferred embodiments, a activin antibody may inhibit activin frombinding to a type I and/or type II receptor (e.g., ActRIIB and/or ALK7)and thus inhibit activin-mediated signaling (e.g., Smad signaling). Insome embodiments, an activin antibody binds to activin B. In someembodiments, an activin antibody binds to activin A. In someembodiments, an activin antibody binds to activin A and activin B. Insome embodiments, an activin antibody binds to activin AB. In someembodiments, an activin antibody binds to activin C. In someembodiments, an activin antibody binds to activin E. In someembodiments, an activin antibody binds to activin A and activin C. Insome embodiments, an activin antibody binds to activin AC. In someembodiments, an activin antibody binds to activin A and activin E. Insome embodiments, an activin antibody binds to activin AE. In someembodiments, an activin antibody binds to activin B and activin C. Insome embodiments, an activin antibody binds to activin BC. In someembodiments, an activin antibody binds to activin B and activin E. Insome embodiments, an activin antibody binds to activin BE. In someembodiments, an activin antibody binds to activin A, activin B, andactivin C. In some embodiments, an activin antibody binds to activin A,activin B, and activin E. Optionally, an activin antibody that binds toone or more of activin A, activin B, and activin C may further bind toactivin E. In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to activin and further binds to, for example, one or moreadditional TGF-β superfamily ligands [e.g., GDF11, GDF8, BMP10, BMP6,BMP5, nodal, and GDF3], one or more type I receptor and/or type IIreceptors (e.g., ActRIIB and/or ALK7), and/or one or more co-receptors.In some embodiments, a multispecific antibody that binds to activin doesnot bind or does not substantially bind to BMP9 (e.g., binds to BMP9with a K_(D) of greater than 1×10⁻⁷ M or has relatively modest binding,e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M. In some embodiments, thedisclosure relates to combinations of antibodies, and uses thereof,wherein the combination of antibodies comprises an activin antibody andone or more additional antibodies that bind to, for example, one or moreadditional TGF-β superfamily ligand [e.g., GDF11, GDF8 GDF3, BMP6,BMP10, nodal, and BMP5], one or more type I receptor and/or type IIreceptors (e.g., ActRIIB and/or ALK7), and/or one or more co-receptors.In some embodiments, a combination of antibodies that comprises anactivin antibody does not comprise a BMP9 antibody.

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least BMP6. Therefore, insome embodiments, an ALK7:ActRIIB antagonist antibody, or combination ofantibodies, binds to at least BMP6. As used herein, a BMP6 antibody (oranti-BMP6 antibody) generally refers to an antibody that can bind toBMP6 with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting BMP6. In certainembodiments, the extent of binding of a BMP6 antibody to an unrelated,non-BMP6 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or less than about 1% of the binding of the antibody to BMP6 asmeasured, for example, by a radioimmunoassay (MA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, aBMP6 antibody binds to an epitope of BMP6 that is conserved among BMP6from different species. In certain preferred embodiments, an anti-BMP6antibody binds to human BMP6. In some embodiments, a BMP6 antibody mayinhibit BMP6 from binding to a type I and/or type II receptor (e.g.,ActRIIB and/or ALK7) and thus inhibit BMP6-mediated signaling (e.g.,Smad signaling). In some embodiments, a BMP6 antibody may inhibit BMP6from binding to a co-receptor and thus inhibit BMP6-mediated signaling(e.g., Smad signaling). In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to BMP6 and further binds to, for example, one or moreadditional TGF-β superfamily ligands [e.g., activin (e.g., activin A,activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE, activin BE), GDF8, BMP10, GDF11, BMP5, nodal, and GDF3], oneor more type I receptor and/or type II receptors (e.g., ActRIIB and/orALK7), and/or one or more co-receptors. In some embodiments, amultispecific antibody that binds to BMP6 does not bind or does notsubstantially bind to BMP9 (e.g., binds to BMP9 with a K_(D) of greaterthan 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M orabout 1×10⁻⁹ M). In some embodiments, the disclosure relates tocombinations of antibodies, and uses thereof, wherein the combination ofantibodies comprises a BMP6 antibody and one or more additionalantibodies that bind to, for example, one or more additional TGF-βsuperfamily ligand [e.g., activin (e.g., activin A, activin B, activinC, activin E, activin AB, activin AC, activin BC, activin AE, activinBE), GDF8 GDF3, GDF11, BMP10, nodal, and BMP5], one or more type Ireceptor and/or type II receptors (e.g., ActRIIB and/or ALK7), and/orone or more co-receptors. In some embodiments, a combination ofantibodies that comprises a BMP6 antibody does not comprise a BMP9antibody.

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least GDF3. Therefore, insome embodiments, an ALK7:ActRIIB antagonist antibody, or combination ofantibodies, binds to at least GDF3. As used herein, a GDF3 antibody (oranti-GDF3 antibody) generally refers to an antibody that binds to GDF3with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting GDF3. In certainembodiments, the extent of binding of a GDF3 antibody to an unrelated,non-GDF3 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or less than about 1% of the binding of the antibody to GDF3 asmeasured, for example, by a radioimmunoassay (MA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, aGDF3 antibody binds to an epitope of GDF3 that is conserved among GDF3from different species. In certain preferred embodiments, a GDF3antibody binds to human GDF3. In some embodiments, a GDF3 antibody mayinhibit GDF3 from binding to a type I and/or type II receptor (e.g.,ActRIIB and/or ALK7) and thus inhibit GDF3-mediated signaling (e.g.,Smad signaling). In some embodiments, a GDF3 antibody may inhibit GDF3from binding to a co-receptor and thus inhibit GDF3-mediated signaling(e.g., Smad signaling). In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to GDF3 and further binds to, for example, one or moreadditional TGF-β superfamily ligands [e.g., activin (e.g., activin A,activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE, activin BE), GDF11, BMP10, BMP6, BMP5, and nodal, one ormore type I receptor and/or type II receptors (e.g., ActRIIB and/orALK7), and/or one or more co-receptors. In some embodiments, amultispecific antibody that binds to GDF3 does not bind or does notsubstantially bind to BMP9 (e.g., binds to BMP9 with a K_(D) of greaterthan 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M orabout 1×10⁻⁹ M). In some embodiments, the disclosure relates tocombinations of antibodies, and uses thereof, wherein the combination ofantibodies comprises a GDF3 antibody and one or more additionalantibodies that bind to, for example, one or more additional TGF-βsuperfamily ligand [e.g., activin (e.g., activin A, activin B, activinC, activin E, activin AB, activin AC, activin BC, activin AE, activinBE), GDF11, BMP10, BMP6, BMP5, and nodal, one or more type I receptorand/or type II receptors (e.g., ActRIIB and/or ALK7), and/or one or moreco-receptors. In some embodiments, a combination of antibodies thatcomprises a GDF3 antibody does not comprise a BMP9 antibody.

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least BMP5. Therefore, insome embodiments, an ALK7:ActRIIB antagonist antibody, or combination ofantibodies, binds to at least BMP5. As used herein, a BMP5 antibody (oranti-BMP5 antibody) generally refers to an antibody that binds to BMP5with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting BMP5. In certainembodiments, the extent of binding of a BMP5 antibody to an unrelated,non-BMP5 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or less than about 1% of the binding of the antibody to BMP5 asmeasured, for example, by a radioimmunoassay (MA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, aBMP5 antibody binds to an epitope of BMP5 that is conserved among BMP5from different species. In certain preferred embodiments, an anti-BMP5antibody binds to human BMP5. In some embodiments, a BMP5 antibody mayinhibit BMP5 from binding to a type I and/or type II receptor (e.g.,ActRIIB and/or ALK7) and thus inhibit BMP5-mediated signaling (e.g.,Smad signaling). In some embodiments, a BMP5 antibody may inhibit BMP5from binding to a co-receptor and thus inhibit BMP5-mediated signaling(e.g., Smad signaling). In some embodiments, the disclosure relates to amultispecific antibody (e.g., bi-specific antibody), and uses thereof,that binds to BMP5 and further binds to, for example, one or moreadditional TGF-β superfamily ligands [e.g., activin (e.g., activin A,activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE and activin BE), GDF8, BMP10, BMP6, GDF11, nodal, and GDF3],one or more type I receptor and/or type II receptors (e.g., ActRIIBand/or ALK7), and one or more co-receptors. In some embodiments, amultispecific antibody that binds to BMP5 does not bind or does notsubstantially bind to BMP9 (e.g., binds to BMP9 with a K_(D) of greaterthan 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M orabout 1×10⁻⁹ M). In some embodiments, the disclosure relates tocombinations of antibodies, and uses thereof, wherein the combination ofantibodies comprises a BMP5 antibody and one or more additionalantibodies that bind to, for example, one or more additional TGF-βsuperfamily ligand [e.g., activin (e.g., activin A, activin B, activinC, activin E, activin AB, activin AC, activin BC, activin AE, andactivin BE), GDF8 GDF3, BMP6, BMP10, nodal, and GDF11], type I receptor,and/or type II receptors (e.g., ActRIIB and/or ALK7), and/orco-receptor. In some embodiments, a combination of antibodies thatcomprises a BMP5 antibody does not comprise a BMP9 antibody.

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least BMP10. Therefore,in some embodiments, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, binds to at least BMP10. As used herein, a BMP10 antibody(or anti-BMP10 antibody) generally refers to an antibody that can bindto BMP10 with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting BMP10. In certainembodiments, the extent of binding of a BMP10 antibody to an unrelated,non-BMP10 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or less than about 1% of the binding of the antibody to BMP10 asmeasured, for example, by a radioimmunoassay (MA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, aBMP10 antibody binds to an epitope of BMP10 that is conserved amongBMP10 from different species. In certain preferred embodiments, ananti-BMP10 antibody binds to human BMP10. In some embodiments, a BMP10antibody may inhibit BMP10 from binding to a type I and/or type IIreceptor (e.g., ActRIIB and/or ALK7) and thus inhibit BMP10-mediatedsignaling (e.g., Smad signaling). In some embodiments, a BMP10 antibodymay inhibit BMP10 from binding to a co-receptor and thus inhibitBMP10-mediated signaling (e.g., Smad signaling). In some embodiments,the disclosure relates to a multispecific antibody (e.g., bi-specificantibody), and uses thereof, that binds to BMP10 and further binds to,for example, one or more additional TGF-β superfamily ligands [e.g.,activin (e.g., activin A, activin B, activin C, activin E, activin AB,activin AC, activin BC, activin AE and activin BE), GDF8, GDF11, BMP6,BMP5, nodal, and GDF3], one or more type I receptor and/or type IIreceptors (e.g., ActRIIB and/or ALK7), and/or one or more co-receptors.In some embodiments, a multispecific antibody that binds to BMP10 doesnot bind or does not substantially bind to BMP9 (e.g., binds to BMP9with a K_(D) of greater than 1×10⁻⁷ M or has relatively modest binding,e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, thedisclosure relates to combinations of antibodies, and uses thereof,wherein the combination of antibodies comprises a BMP10 antibody and oneor more additional antibodies that bind to, for example, one or moreadditional TGF-β superfamily ligand [e.g., activin (e.g., activin A,activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE and activin BE), GDF8 GDF3, BMP6, GDF11, nodal, and BMP5],one or more type I receptor and/or type II receptors (e.g., ActRIIBand/or ALK7), and/or one or more co-receptors. In some embodiments, acombination of antibodies that comprises a BMP10 antibody does notcomprise a BMP9 antibody.

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least nodal. Therefore,in some embodiments, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, binds to at least nodal. As used herein, a nodal antibody(or anti-nodal antibody) generally refers to an antibody that can bindto nodal with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting nodal. In certainembodiments, the extent of binding of a nodal antibody to an unrelated,non-nodal protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or less than about 1% of the binding of the antibody to nodal asmeasured, for example, by a radioimmunoassay (MA), Biacore, or otherprotein interaction or binding affinity assay. In certain embodiments, anodal antibody binds to an epitope of nodal that is conserved amongnodal from different species. In certain preferred embodiments, ananti-nodal antibody binds to human nodal. In some embodiments, a nodalantibody may inhibit nodal from binding to a type I and/or type IIreceptor (e.g., ActRIIB and/or ALK7) and thus inhibit nodal-mediatedsignaling (e.g., Smad signaling). In some embodiments, a nodal antibodymay inhibit nodal from binding to a co-receptor and thus inhibitnodal-mediated signaling (e.g., Smad signaling). In some embodiments,the disclosure relates to a multispecific antibody (e.g., bi-specificantibody), and uses thereof, that binds to nodal and further binds to,for example, one or more additional TGF-β superfamily ligands [e.g.,activin (e.g., activin A, activin B, activin C, activin E, activin AB,activin AC, activin BC, activin AE and activin BE), GDF8, GDF11, BMP6,BMP5, BMP10, and GDF3], one or more type I receptor and/or type IIreceptors (e.g., ActRIIB and/or ALK7), and/or one or more co-receptors.In some embodiments, a multispecific antibody that binds to nodal doesnot bind or does not substantially bind to BMP9 (e.g., binds to BMP9with a K_(D) of greater than 1×10⁻⁷ M or has relatively modest binding,e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, thedisclosure relates to combinations of antibodies, and uses thereof,wherein the combination of antibodies comprises a nodal antibody and oneor more additional antibodies that bind to, for example, one or moreadditional TGF-β superfamily ligand [e.g., activin (e.g., activin A,activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE and activin BE), GDF8 GDF3, BMP6, GDF11, BMP10, and BMP5],one or more type I receptor and/or type II receptors (e.g., ActRIIBand/or ALK7), and/or one or more co-receptors. In some embodiments, acombination of antibodies that comprises a nodal antibody does notcomprise a BMP9 antibody.

With respect to antibodies that bind to and antagonize ligands that bindto ALK7:ActRIIB, [e.g., activin (e.g., activin A, activin B, activin C,activin E, activin AB, activin AC, activin BC, activin AE and activinBE), GDF8 GDF3, BMP6, GDF11, BMP10, and BMP5], it is contemplated thatan antibody may be designed as a bispecific antibody comprising a firstportion that binds to an epitope of such ligand, such that the firstportion of the antibody competes for binding with a type I receptor andcomprising a second portion that binds to an epitope of such ligand,such that the second portion of the antibody competes for binding with atype II receptor. In this manner, a bispecific antibody targeting asingle ligand can be designed to mimic the dual type I-type II receptorbinding blockade that may be conferred by an ALK7:ActRIIBheteromultimer. Similarly it is contemplated that the same effect couldbe achieved using a combination of two or more antibodies wherein atleast a first antibody binds to an epitope of such ligand, such that thefirst antibody competes for binding with a type I receptor and at leasta second antibody binds to an epitope of such ligand, such that thesecond antibody competes for binding with a type II receptor.

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least ActRIIB Therefore,in some embodiments, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, binds to at least ActRIIB As used herein, an ActRIIBantibody (anti-ActRIIB antibody) generally refers to an antibody thatbinds to ActRIIB with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting ActRIIB Incertain embodiments, the extent of binding of an anti-ActRIIB antibodyto an unrelated, non-ActRIIB protein is less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibodyto ActRIIB as measured, for example, by a radioimmunoassay (RIA),Biacore, or other protein-protein interaction or binding affinity assay.In certain embodiments, an anti-ActRIIB antibody binds to an epitope ofActRIIB that is conserved among ActRIIB from different species. Incertain preferred embodiments, an anti-ActRIIB antibody binds to humanActRIIB In some embodiments, an anti-ActRIIB antibody may inhibit one ormore TGF-β superfamily ligands [e.g., GDF8, activin (e.g., activin A,activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE and activin BE) GDF3, BMP6, BMP10, nodal and BMP9] frombinding to ActRIIB and/or ALK7. In some embodiments, an anti-ActRIIBantibody is a multispecific antibody (e.g., bi-specific antibody) thatbinds to ActRIIB and one or more TGF-β superfamily ligands [e.g., GDF11,GDF8, activin (e.g., activin A, activin B, activin C, activin E, activinAB, activin AC) GDF3, BMP6, BMP10, nodal, and BMP9], type I receptor(e.g., ALK7), co-receptor, and/or an additional type II receptor. Insome embodiments, the disclosure relates to combinations of antibodies,and uses thereof, wherein the combination of antibodies comprises ananti-ActRIIB antibody and one or more additional antibodies that bindto, for example, one or more TGF-β superfamily ligands [e.g., GDF11,GDF8, activin (e.g., activin A, activin B, activin C, activin E, activinAB, activin AC, activin BC, activin AE and activin BE) GDF3, BMP6,BMP10, nodal, and BMP9], co-receptors, type I receptors (e.g., ALK7),and/or additional type II receptors. It should be noted that ActRIIB hassequence similarity to ActRIIA and therefore antibodies that bind toActRIIB, in some instances, may also bind to and/or inhibit ActRIIA.

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least ALK7. Therefore, insome embodiments, an ALK7:ActRIIB antagonist antibody, or combination ofantibodies, binds to at least ALK7. As used herein, an ALK7 antibody(anti-ALK7 antibody) generally refers to an antibody that binds to ALK7with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting ALK7. In certainembodiments, the extent of binding of an anti-ALK7 antibody to anunrelated, non-ALK7 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or less than about 1% of the binding of the antibody to ALK7as measured, for example, by a radioimmunoassay (MA), Biacore, or otherprotein-protein interaction or binding affinity assay. In certainembodiments, an anti-ALK7 antibody binds to an epitope of ALK7 that isconserved among ALK7 from different species. In certain preferredembodiments, an anti-ALK7 antibody binds to human ALK7. In someembodiments, an anti-ALK7 antibody may inhibit one or more TGF-βsuperfamily ligands [e.g., GDF11, GDF8, activin (e.g., activin A,activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE and activin BE) GDF3, BMP5, BMP6, and BMP10] from binding toa type I receptor (e.g., ALK7), type II receptor (e.g., ActRIIB), orco-receptor. In some embodiments, an anti-ALK7 antibody is amultispecific antibody (e.g., bi-specific antibody) that binds to ALK7and one or more TGF-β superfamily ligands [e.g., activin (e.g., activinA, activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE and activin BE), GDF11, GDF8, BMP10, BMP6, BMP5, nodal, andGDF3, type II receptors (e.g., ActRIIB), co-receptors, and/or anadditional type I receptor. In some embodiments, the disclosure relatesto combinations of antibodies, and uses thereof, wherein the combinationof antibodies comprises an anti-ALK7 antibody and one or more additionalantibodies that bind to, for example, one or more TGF-β superfamilyligands [e.g., activin (e.g., activin A, activin B, activin C, activinE, activin AB, activin AC, activin BC, activin AE and activin BE),GDF11, GDF8, BMP10, BMP6, BMP5, nodal, and GDF3], co-receptors, anadditional type I receptor, and/or type II receptors (e.g., ActRIIB)

In certain aspects, an ALK7:ActRIIB antagonist antibody, or combinationof antibodies, is an antibody that inhibits at least Cripto or Cryptic.Therefore, in some embodiments, an ALK7:ActRIIB antagonist antibody, orcombination of antibodies, binds to at least Cripto or Cryptic. As usedherein, an Cripto antibody (anti-Cripto antibody) generally refers to anantibody that binds to Cripto with sufficient affinity such that theantibody is useful as a diagnostic and/or therapeutic agent in targetingCripto. In certain embodiments, the extent of binding of an anti-Criptoantibody to an unrelated, non-Cripto protein is less than about 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of theantibody to Cripto as measured, for example, by a radioimmunoassay (MA),Biacore, or other protein-protein interaction or binding affinity assay.In certain embodiments, an anti-Cripto antibody binds to an epitope ofCripto that is conserved among Cripto from different species. In certainpreferred embodiments, an anti-Cripto antibody binds to human Cripto. Asused herein, a Cryptic antibody (anti-Cryptic antibody) generally refersto an antibody that binds to Cryptic with sufficient affinity such thatthe antibody is useful as a diagnostic and/or therapeutic agent intargeting Cryptic. In certain embodiments, the extent of binding of ananti-Cripto antibody to an unrelated, non-Cryptic protein is less thanabout 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of thebinding of the antibody to Cryptic as measured, for example, by aradioimmunoassay (MA), Biacore, or other protein-protein interaction orbinding affinity assay. In certain embodiments, an anti-Cryptic antibodybinds to an epitope of Cryptic that is conserved among Cryptic fromdifferent species. In certain preferred embodiments, an anti-Crypticantibody binds to human Cryptic. In some embodiments, an anti-Cripto orCryptic antibody may inhibit one or more TGF-β superfamily ligands[e.g., activin (e.g., activin A, activin B, activin C, activin E,activin AB, activin AC, activin BC, activin AE and activin BE), GDF11,GDF8, BMP10, BMP6, BMP5, nodal, and GDF3] from binding to Cripto orCryptic, respectively, or to ActRIIB and/or ALK7. In some embodiments,an anti-Cripto or Cryptic antibody may inhibit nodal from binding to atype I and/or type II receptor. In some embodiments, an anti-Cripto orCryptic antibody is a multispecific antibody (e.g., bi-specificantibody) that binds to Cripto or Cryptic and one or more TGF-βsuperfamily [e.g., activin (e.g., activin A, activin B, activin C,activin E, activin AB, activin AC, activin BC, activin AE and activinBE), GDF11, GDF8, BMP10, BMP6, BMP5, nodal, and GDF3], type I receptor(e.g., ALK7), additional co-receptor, and/or type II receptor. In someembodiments an antibody may bind to both Cripto and Cryptic. In someembodiments, the disclosure relates to combinations of antibodies, anduses thereof, wherein the combination of antibodies comprises ananti-Cripto or Cryptic antibody and one or more additional antibodiesthat bind to, for example, one or more TGF-β superfamily ligands [e.g.,activin (e.g., activin A, activin B, activin C, activin E, activin AB,activin AC, activin BC, activin AE and activin BE), GDF11, GDF8, BMP10,BMP6, BMP5, nodal, and GDF3], additional co-receptors, type I receptors(e.g., ALK7), and/or type II receptors.

As described herein, there are a variety of methods for generatingheteromultimeric complexes. Such methods may be used to generateheteromultimer complexes comprising an antibody-binding domain (e.g., acomplex of V_(L) and V_(H) chains) and one or more polypeptides selectedfrom an ALK7 polypeptide, an ActRIIB polypeptide, an ALK7:ActRIIBheteromer, or an ALK7:ActRIIB single trap polypeptide. See FIGS. 10A,10B and 12D. For example, in some embodiments, the disclosure providesprotein complexes comprising a ligand-binding domain of an antibody thatbinds to an ALK7:ActRIIB-binding ligand [e.g., activin (e.g., activin A,activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE and activin BE), GDF11, GDF8, BMP10, BMP6, BMP5, nodal, andGDF3] which is covalently or non-covalently associated with an ALK7polypeptide. In some embodiments, the disclosure provides proteincomplexes comprising a ligand-binding domain of an antibody that bindsto an ALK7:ActRIIB-binding ligand [e.g., activin (e.g., activin A,activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE and activin BE), GDF11, GDF8, BMP10, BMP6, BMP5, nodal, andGDF3] which is covalently or non-covalently associated with an ActRIIBpolypeptide. In some embodiments, the disclosure provides proteincomplexes comprising a ligand-binding domain of an antibody that bindsto an ALK7:ActRIIB-binding ligand [e.g., activin (e.g., activin A,activin B, activin C, activin E, activin AB, activin AC, activin BC,activin AE and activin BE), GDF11, GDF8, BMP10, BMP6, BMP5, nodal, andGDF3] which is covalently or non-covalently associated with anALK7:ActRIIB single-chain ligand trap. In some embodiments, thedisclosure provides protein complexes comprising a ligand-binding domainof an antibody that binds to an ALK7:ActRIIB-binding ligand [e.g.,activin (e.g., activin A, activin B, activin C, activin E, activin AB,activin AC, activin BC, activin AE and activin BE), GDF11, GDF8, BMP10,BMP6, BMP5, nodal, and GDF3] which is covalently or non-covalentlyassociated with ALK7:ActRIIB heteromultimer.

The term antibody is used herein in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity. An antibody fragment refers to amolecule other than an intact antibody that comprises a portion of anintact antibody that binds the antigen to which the intact antibodybinds. Examples of antibody fragments include, but are not limited to,Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linear antibodies;single-chain antibody molecules (e.g., scFv); and multispecificantibodies formed from antibody fragments [see, e.g., Hudson et al.(2003) Nat. Med. 9:129-134; Plückthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, NewYork), pp. 269-315 (1994); WO 93/16185; and U.S. Pat. Nos. 5,571,894;5,587,458; and 5,869,046]. Diabodies are antibody fragments with twoantigen-binding sites that may be bivalent or bispecific [see, e.g., EP404,097; WO 1993/01161; Hudson et al. (2003) Nat. Med. 9:129-134 (2003);and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448].Triabodies and tetrabodies are also described in Hudson et al. (2003)Nat. Med. 9:129-134. Single-domain antibodies are antibody fragmentscomprising all or a portion of the heavy-chain variable domain or all ora portion of the light-chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody[see, e.g., U.S. Pat. No. 6,248,516]. Antibodies disclosed herein may bepolyclonal antibodies or monoclonal antibodies. In certain embodiments,the antibodies of the present disclosure comprise a label attachedthereto and able to be detected (e.g., the label can be a radioisotope,fluorescent compound, enzyme, or enzyme co-factor). In certain preferredembodiments, the antibodies of the present disclosure are isolatedantibodies. In certain preferred embodiments, the antibodies of thepresent disclosure are recombinant antibodies.

The antibodies herein may be of any class. The class of an antibodyrefers to the type of constant domain or constant region possessed byits heavy chain. There are five major classes of antibodies: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), for example, IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, andIgA₂. The heavy chain constant domains that correspond to the differentclasses of immunoglobulins are called alpha, delta, epsilon, gamma, andmu.

In general, an antibody for use in the methods disclosed hereinspecifically binds to its target antigen, preferably with high bindingaffinity. Affinity may be expressed as a K_(D) value and reflects theintrinsic binding affinity (e.g., with minimized avidity effects).Typically, binding affinity is measured in vitro, whether in a cell-freeor cell-associated setting. Any of a number of assays known in the art,including those disclosed herein, can be used to obtain binding affinitymeasurements including, for example, Biacore, radiolabeledantigen-binding assay (RIA), and ELISA. In some embodiments, antibodiesof the present disclosure bind to their target antigens (e.g. ALK7,ActRIIB, activin, GDF11, GDF8, BMP10, BMP6, GDF3, nodal, and/or BMP5)with at least a K_(D) of 1×10⁻⁷ or stronger, 1×10⁻⁸ or stronger, 1×10⁻⁹or stronger, 1×10⁻¹⁰ or stronger, 1×10⁻¹¹ or stronger, 1×10⁻¹² orstronger, 1×10⁻¹³ or stronger, or 1×10⁻¹⁴ or stronger.

In certain embodiments, K_(D) is measured by RIA performed with the Fabversion of an antibody of interest and its target antigen as describedby the following assay. Solution binding affinity of Fabs for theantigen is measured by equilibrating Fab with a minimal concentration ofradiolabeled antigen (e.g., ¹²⁵I-labeled) in the presence of a titrationseries of unlabeled antigen, then capturing bound antigen with ananti-Fab antibody-coated plate [see, e.g., Chen et al. (1999) J. Mol.Biol. 293:865-881]. To establish conditions for the assay, multi-wellplates (e.g., MICROTITER® from Thermo Scientific) are coated (e.g.,overnight) with a capturing anti-Fab antibody (e.g., from Cappel Labs)and subsequently blocked with bovine serum albumin, preferably at roomtemperature (approximately 23° C.). In a non-adsorbent plate,radiolabeled antigen are mixed with serial dilutions of a Fab ofinterest [e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599]. The Fab ofinterest is then incubated, preferably overnight but the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation, preferably at room temperature for aboutone hour. The solution is then removed and the plate is washed timesseveral times, preferably with polysorbate 20 and PBS mixture. When theplates have dried, scintillant (e.g., MICROSCINT® from Packard) isadded, and the plates are counted on a gamma counter (e.g., TOPCOUNT®from Packard).

According to another embodiment, K_(D) is measured using surface plasmonresonance assays using, for example a BIACORE® 2000 or a BIACORE® 3000(BIAcore, Inc., Piscataway, N.J.) with immobilized antigen CMS chips atabout 10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CMS, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NETS) according to the supplier's instructions.For example, an antigen can be diluted with 10 mM sodium acetate, pH4.8, to 5 μg/ml (about 0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at a flow rate ofapproximately 25 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using, for example, a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on) [see, e.g., Chen et al., (1999) J. Mol. Biol.293:865-881]. If the on-rate exceeds, for example, 10⁶M⁻¹ s⁻¹ by thesurface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (e.g.,excitation=295 nm; emission=340 nm, 16 nm band-pass) of a 20 nManti-antigen antibody (Fab form) in PBS in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-AMINCO® spectrophotometer (ThermoSpectronic) with a stirred cuvette.

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein. The nucleic acid and amino acid sequences of humanActRIIB, activin (activin A, activin B, activin C, and activin E),GDF11, GDF8, BMP10, BMP6, GDF3, nodal, and/or BMP5 are known in the art.In addition, numerous methods for generating antibodies are well knownin the art, some of which are described herein. Therefore antibodyantagonists for use in accordance with this disclosure may be routinelymade by the skilled person in the art based on the knowledge in the artand teachings provided herein.

In certain embodiments, an antibody provided herein is a chimericantibody. A chimeric antibody refers to an antibody in which a portionof the heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species. Certain chimeric antibodies aredescribed, for example, in U.S. Pat. No. 4,816,567; and Morrison et al.,(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855. In some embodiments, achimeric antibody comprises a non-human variable region (e.g., avariable region derived from a mouse, rat, hamster, rabbit, or non-humanprimate, such as a monkey) and a human constant region. In someembodiments, a chimeric antibody is a “class switched” antibody in whichthe class or subclass has been changed from that of the parent antibody.In general, chimeric antibodies include antigen-binding fragmentsthereof.

In certain embodiments, a chimeric antibody provided herein is ahumanized antibody. A humanized antibody refers to a chimeric antibodycomprising amino acid residues from non-human hypervariable regions(HVRs) and amino acid residues from human framework regions (FRs). Incertain embodiments, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the HVRs (e.g., CDRs) correspond to those of anon-human antibody, and all or substantially all of the FRs correspondto those of a human antibody. A humanized antibody optionally maycomprise at least a portion of an antibody constant region derived froma human antibody. A “humanized form” of an antibody, e.g., a non-humanantibody, refers to an antibody that has undergone humanization.Humanized antibodies and methods of making them are reviewed, forexample, in Almagro and Fransson (2008) Front. Biosci. 13:1619-1633 andare further described, for example, in Riechmann et al., (1988) Nature332:323-329; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA86:10029-10033; U.S. Pat. Nos. 5,821,337; 7,527,791; 6,982,321; and7,087,409; Kashmiri et al., (2005) Methods 36:25-34 [describing SDR(a-CDR) grafting]; Padlan, Mol. Immunol. (1991) 28:489-498 (describing“resurfacing”); Dall'Acqua et al. (2005) Methods 36:43-60 (describing“FR shuffling”); Osbourn et al. (2005) Methods 36:61-68; and Klimka etal. Br. J. Cancer (2000) 83:252-260 (describing the “guided selection”approach to FR shuffling). Human framework regions that may be used forhumanization include but are not limited to: framework regions selectedusing the “best-fit” method [see, e.g., Sims et al. (1993) J. Immunol.151:2296]; framework regions derived from the consensus sequence ofhuman antibodies of a particular subgroup of light or heavy chainvariable regions [see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci.USA, 89:4285; and Presta et al. (1993) J. Immunol., 151:2623]; humanmature (somatically mutated) framework regions or human germlineframework regions [see, e.g., Almagro and Fransson (2008) Front. Biosci.13:1619-1633]; and framework regions derived from screening FR libraries[see, e.g., Baca et al., (1997) J. Biol. Chem. 272:10678-10684; andRosok et al., (1996) J. Biol. Chem. 271:22611-22618].

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel (2008) Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr.Opin. Immunol. 20:450-459. For example, human antibodies may be preparedby administering an immunogen (e.g., a GDF11 polypeptide, an activin Bpolypeptide, an ActRIIA polypeptide, or an ActRIIB polypeptide) to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicanimals, the endogenous immunoglobulin loci have generally beeninactivated. For a review of methods for obtaining human antibodies fromtransgenic animals see, for example, Lonberg (2005) Nat. Biotech.23:1117-1125; U.S. Pat. Nos. 6,075,181 and 6,150,584 (describingXENOMOUSE™ technology); U.S. Pat. No. 5,770,429 (describing HuMab®technology); U.S. Pat. No. 7,041,870 (describing K-M MOUSE® technology);and U.S. Patent Application Publication No. 2007/0061900 (describingVelociMouse® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, for example, bycombining with a different human constant region.

Human antibodies provided herein can also be made by hybridoma-basedmethods. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described [see,e.g., Kozbor J. Immunol., (1984) 133: 3001; Brodeur et al. (1987)Monoclonal Antibody Production Techniques and Applications, pp. 51-63,Marcel Dekker, Inc., New York; and Boerner et al. (1991) J. Immunol.,147: 86]. Human antibodies generated via human B-cell hybridomatechnology are also described in Li et al., (2006) Proc. Natl. Acad.Sci. USA, 103:3557-3562. Additional methods include those described, forexample, in U.S. Pat. No. 7,189,826 (describing production of monoclonalhuman IgM antibodies from hybridoma cell lines) and Ni, XiandaiMianyixue (2006) 26(4):265-268 (2006) (describing human-humanhybridomas). Human hybridoma technology (Trioma technology) is alsodescribed in Vollmers and Brandlein (2005) Histol. Histopathol.,20(3):927-937 (2005) and Vollmers and Brandlein (2005) Methods Find Exp.Clin. Pharmacol., 27(3):185-91. Human antibodies provided herein mayalso be generated by isolating Fv clone variable-domain sequencesselected from human-derived phage display libraries. Suchvariable-domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are known in the art and described herein.

For example, antibodies of the present disclosure may be isolated byscreening combinatorial libraries for antibodies with the desiredactivity or activities. A variety of methods are known in the art forgenerating phage display libraries and screening such libraries forantibodies possessing the desired binding characteristics. Such methodsare reviewed, for example, in Hoogenboom et al. (2001) in Methods inMolecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa,N.J. and further described, for example, in the McCafferty et al. (1991)Nature 348:552-554; Clackson et al., (1991) Nature 352: 624-628; Markset al. (1992) J. Mol. Biol. 222:581-597; Marks and Bradbury (2003) inMethods in Molecular Biology 248:161-175, Lo, ed., Human Press, Totowa,N.J.; Sidhu et al. (2004) J. Mol. Biol. 338(2):299-310; Lee et al.(2004) J. Mol. Biol. 340(5):1073-1093; Fellouse (2004) Proc. Natl. Acad.Sci. USA 101(34):12467-12472; and Lee et al. (2004) J. Immunol. Methods284(1-2): 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al. (1994) Ann. Rev.Immunol., 12: 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen (e.g., ALK7, ActRIIB, activin, GDF11, GDF8, BMP10, BMP6, GDF3,and/or BMP5) without the requirement of constructing hybridomas.Alternatively, the naive repertoire can be cloned (e.g., from human) toprovide a single source of antibodies to a wide range of non-self andalso self-antigens without any immunization as described by Griffiths etal. (1993) EMBO J, 12: 725-734. Finally, naive libraries can also bemade synthetically by cloning unrearranged V-gene segments from stemcells, and using PCR primers containing random sequence to encode thehighly variable CDR3 regions and to accomplish rearrangement in vitro,as described by Hoogenboom and Winter (1992) J. Mol. Biol., 227:381-388. Patent publications describing human antibody phage librariesinclude, for example: U.S. Pat. No. 5,750,373, and U.S. PatentPublication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126,2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

In certain embodiments, an antibody provided herein is a multispecificantibody, for example, a bispecific antibody. Multispecific antibodies(typically monoclonal antibodies) that have binding specificities for atleast two different epitopes (e.g., two, three, four, five, or six ormore) on one or more (e.g., two, three, four, five, six or more)antigens.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulinheavy-chain/light-chain pairs having different specificities [see, e.g.,Milstein and Cuello (1983) Nature 305: 537; International patentpublication no. WO 93/08829; and Traunecker et al. (1991) EMBO J. 10:3655, and U.S. Pat. No. 5,731,168 (“knob-in-hole” engineering)].Multispecific antibodies may also be made by engineering electrostaticsteering effects for making antibody Fc-heterodimeric molecules (see,e.g., WO 2009/089004A1); cross-linking two or more antibodies orfragments [see, e.g., U.S. Pat. No. 4,676,980; and Brennan et al. (1985)Science, 229: 81]; using leucine zippers to produce bispecificantibodies [see, e.g., Kostelny et al. (1992) J. Immunol.,148(5):1547-1553]; using “diabody” technology for making bispecificantibody fragments [see, e.g., Hollinger et al. (1993) Proc. Natl. Acad.Sci. USA, 90:6444-6448]; using single-chain Fv (sFv) dimers [see, e.g.,Gruber et al. (1994) J. Immunol., 152:5368]; and preparing trispecificantibodies (see, e.g., Tutt et al. (1991) J. Immunol. 147: 60.Multispecific antibodies can be prepared as full-length antibodies orantibody fragments. Engineered antibodies with three or more functionalantigen-binding sites, including “Octopus antibodies,” are also includedherein [see, e.g., US 2006/0025576A1].

In certain embodiments, an antibody disclosed herein is a monoclonalantibody. Monoclonal antibody refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical and/or bind the sameepitope, except for possible variant antibodies, e.g., containingnaturally occurring mutations or arising during production of amonoclonal antibody preparation, such variants generally being presentin minor amounts. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentepitopes, each monoclonal antibody of a monoclonal antibody preparationis directed against a single epitope on an antigen. Thus, the modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present methods may be made by a variety of techniques,including but not limited to the hybridoma method, recombinant DNAmethods, phage-display methods, and methods utilizing transgenic animalscontaining all or part of the human immunoglobulin loci, such methodsand other exemplary methods for making monoclonal antibodies beingdescribed herein.

For example, by using immunogens derived from activin,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeby standard protocols [see, e.g., Antibodies: A Laboratory Manual ed. byHarlow and Lane (1988) Cold Spring Harbor Press: 1988]. A mammal, suchas a mouse, hamster, or rabbit, can be immunized with an immunogenicform of the activin polypeptide, an antigenic fragment which is capableof eliciting an antibody response, or a fusion protein. Techniques forconferring immunogenicity on a protein or peptide include conjugation tocarriers or other techniques well known in the art. An immunogenicportion of a activin polypeptide can be administered in the presence ofadjuvant. The progress of immunization can be monitored by detection ofantibody titers in plasma or serum. Standard ELISA or other immunoassayscan be used with the immunogen as antigen to assess the levels ofantibody production and/or level of binding affinity.

Following immunization of an animal with an antigenic preparation ofactivin, antisera can be obtained and, if desired, polyclonal antibodiescan be isolated from the serum. To produce monoclonal antibodies,antibody-producing cells (lymphocytes) can be harvested from animmunized animal and fused by standard somatic cell fusion procedureswith immortalizing cells such as myeloma cells to yield hybridoma cells.Such techniques are well known in the art, and include, for example, thehybridoma technique [see, e.g., Kohler and Milstein (1975) Nature, 256:495-497], the human B cell hybridoma technique [see, e.g., Kozbar et al.(1983) Immunology Today, 4:72], and the EBV-hybridoma technique toproduce human monoclonal antibodies [Cole et al. (1985) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96]. Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with a activin polypeptide, and monoclonalantibodies isolated from a culture comprising such hybridoma cells.

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g., a substitution,deletion, and/or addition) at one or more amino acid positions.

For example, the present disclosure contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions [e.g.,complement-dependent cytotoxicity (CDC) and antibody-dependent cellularcytotoxicity (ADCC)] are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in, forexample, Ravetch and Kinet (1991) Annu. Rev. Immunol. 9:457-492.Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest are described in U.S. Pat. No. 5,500,362;Hellstrom, I. et al. (1986) Proc. Natl. Acad. Sci. USA 83:7059-7063];Hellstrom, I et al. (1985) Proc. Natl. Acad. Sci. USA 82:1499-1502; U.S.Pat. No. 5,821,337; Bruggemann, M. et al. (1987) J. Exp. Med.166:1351-1361. Alternatively, non-radioactive assays methods may beemployed (e.g., ACTI™, non-radioactive cytotoxicity assay for flowcytometry; CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay, Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and natural killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, for example, in an animal model such as that disclosed inClynes et al. (1998) Proc. Natl. Acad. Sci. USA 95:652-656. C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity [see, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402]. To assess complementactivation, a CDC assay may be performed [see, e.g, Gazzano-Santoro etal. (1996) J. Immunol. Methods 202:163; Cragg, M. S. et al. (2003) Blood101:1045-1052; and Cragg, M. S, and M. J. Glennie (2004) Blood103:2738-2743]. FcRn binding and in vivo clearance/half-lifedeterminations can also be performed using methods known in the art[see, e.g., Petkova, S. B. et al. (2006) Intl. Immunol.18(12):1759-1769]. Antibodies of the present disclosure with reducedeffector function include those with substitution of one or more of Fcregion residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants with substitutions at twoor more of amino acid positions 265, 269, 270, 297 and 327, includingthe so-called “DANA” Fc mutant with substitution of residues 265 and 297to alanine (U.S. Pat. No. 7,332,581).

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, for example, inU.S. Pat. No. 7,521,541.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionsbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore binding assay, Biacore AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Maryland), western blots,immunoprecipitation assays, and immunohistochemistry.

In certain embodiments, amino acid sequence variants of the antibodiesand/or the binding polypeptides provided herein are contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the antibody and/or binding polypeptide.Amino acid sequence variants of an antibody and/or binding polypeptidesmay be prepared by introducing appropriate modifications into thenucleotide sequence encoding the antibody and/or binding polypeptide, orby peptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of residues within theamino acid sequences of the antibody and/or binding polypeptide. Anycombination of deletion, insertion, and substitution can be made toarrive at the final construct, provided that the final constructpossesses the desired characteristics, e.g., target-binding (e.g., andactivin such as activin E and/or activin C binding).

Alterations (e.g., substitutions) may be made in HVRs, for example, toimprove antibody affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation athigh frequency during the somatic maturation process [see, e.g.,Chowdhury (2008) Methods Mol. Biol. 207:179-196 (2008)], and/or SDRs(a-CDRs), with the resulting variant VH or VL being tested for bindingaffinity. Affinity maturation by constructing and reselecting fromsecondary libraries has been described in the art [see, e.g., Hoogenboomet al., in Methods in Molecular Biology 178:1-37, O'Brien et al., ed.,Human Press, Totowa, N.J., (2001). In some embodiments of affinitymaturation, diversity is introduced into the variable genes chosen formaturation by any of a variety of methods (e.g., error-prone PCR, chainshuffling, or oligonucleotide-directed mutagenesis). A secondary libraryis then created. The library is then screened to identify any antibodyvariants with the desired affinity. Another method to introducediversity involves HVR-directed approaches, in which several HVRresidues (e.g., 4-6 residues at a time) are randomized. HVR residuesinvolved in antigen binding may be specifically identified, e.g., usingalanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 inparticular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind to the antigen.For example, conservative alterations (e.g., conservative substitutionsas provided herein) that do not substantially reduce binding affinitymay be made in HVRs. Such alterations may be outside of HVR “hotspots”or SDRs. In certain embodiments of the variant VH and VL sequencesprovided above, each HVR either is unaltered, or contains no more thanone, two or three amino acid substitutions.

A useful method for identification of residues or regions of theantibody and/or the binding polypeptide that may be targeted formutagenesis is called “alanine scanning mutagenesis” as described byCunningham and Wells (1989) Science, 244:1081-1085. In this method, aresidue or group of target residues (e.g., charged residues such as Arg,Asp, His, Lys, and Glu) are identified and replaced by a neutral ornegatively charged amino acid (e.g., alanine or polyalanine) todetermine whether the interaction of the antibody-antigen is affected.Further substitutions may be introduced at the amino acid locationsdemonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of anantigen-antibody complex is determined to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues may be targeted or eliminated as candidates for substitution.Variants may be screened to determine whether they contain the desiredproperties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion of the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

In certain embodiments, an antibody and/or binding polypeptide providedherein may be further modified to contain additional nonproteinaceousmoieties that are known in the art and readily available. The moietiessuitable for derivatization of the antibody and/or binding polypeptideinclude but are not limited to water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, polpropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody and/orbinding polypeptide may vary, and if more than one polymer are attached,they can be the same or different molecules. In general, the numberand/or type of polymers used for derivatization can be determined basedon considerations including, but not limited to, the particularproperties or functions of the antibody and/or binding polypeptide to beimproved, whether the antibody derivative and/or binding polypeptidederivative will be used in a therapy under defined conditions.

D. Small Molecule Antagonists

In other aspects, an ALK7:ActRIIB antagonist is a small molecule(ALK7:ActRIIB small molecule antagonist), or combination of smallmolecule antagonists. An ALK7:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, may inhibit, for example, oneor more ALK7:ActRIIB-binding ligands, a type I receptor (e.g., ALK7), atype II receptor (e.g., ActRIIB), and/or co-receptor (e.g., Cripto orCryptic). In some embodiments, ALK7:ActRIIB small molecule antagonist,or combination of small molecule antagonists, inhibits signalingmediated by one or more ALK7:ActRIIB-binding ligands, for example, asdetermined in a cell-based assay such as those described herein. Asdescribed herein, ALK7:ActRIIB small molecule antagonists may be used,alone or in combination with one or more supportive therapies or activeagents, to treat a patient in need thereof (e.g., patients having kidneydisease and/or a metabolic disorder).

In some embodiments, an ALK7:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, inhibits at least GDF11. Insome embodiments, an ALK7:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, inhibits at least GDF8. Insome embodiments, an ALK7:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, inhibits at least activin(activin A, activin B, activin C, activin E, activin AB, activin AC,activin BC, activin AE and/or activin BE). In some embodiments, anALK7:ActRIIB small molecule antagonist, or combination of small moleculeantagonists, inhibits at least GDF11, GDF8, and activin. In someembodiments, an ALK7:ActRIIB small molecule antagonist, or combinationof small molecule antagonists, inhibits at least ALK7. In someembodiments, an ALK7:ActRIIB small molecule antagonist, or combinationof small molecule antagonists, inhibits at least ActRIIB In someembodiments, an ALK7:ActRIIB small molecule antagonist, or combinationof small molecule antagonists, inhibits at least Cripto or Cryptic. Insome embodiments, an ALK7:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, inhibits at least BMP6. Insome embodiments, an ALK7:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, inhibits at least GDF3. Insome embodiments, an ALK7:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, inhibits at least BMP5. Insome embodiments, an ALK7:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, inhibits at least BMP10. Insome embodiments, an ALK7:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, inhibits at least nodal. Insome embodiments, an ALK7:ActRIIB small molecule antagonist, orcombination of small molecule antagonists, as disclosed herein does notinhibit or does not substantially inhibit BMP9.

ALK7:ActRIIB small molecule antagonists can be direct or indirectinhibitors. For example, an indirect small molecule antagonist, orcombination of small molecule antagonists, may inhibit the expression(e.g., transcription, translation, cellular secretion, or combinationsthereof) of at least one or more TGF-β superfamily ligands [e.g.,activin (e.g., activin A, activin B, activin C, activin E, activin AB,activin AC, activin B, activin BC, activin AE, or Activin BE), GDF11,BMP10, BMP9, BMP6, BMP5, GDF3, activin C, activin E, Activin AC, GDF8,BMP10, BMP6, BMP5, nodal, and GDF3, type I receptor (e.g., ALK7), typeII receptors (e.g., ActRIIB), co-receptor (e.g., Cripto or Cryptic),and/or one or more downstream signaling components (e.g., Smads).Alternatively, a direct small molecule antagonist, or combination ofsmall molecule antagonists, may directly bind to and inhibit, forexample, one or more TGF-β superfamily ligands [e.g., activin (e.g.,activin A, activin B, activin C, activin E, activin AB, activin AC,activin B, activin BC, activin AE, or Activin BE), GDF11, BMP10, BMP9,BMP6, BMP5, GDF3, activin C, activin E, activin AC, GDF8, BMP10, BMP6,BMP5, nodal, and GDF3, type I receptors (e.g., ALK7), type II receptors(e.g., ActRIIB), co-receptors (e.g., Cripto or Cryptic), and/ordownstream signaling components (e.g., Smads). Combinations of one ormore indirect and one or more direct ALK7:ActRIIB small moleculeantagonists may be used in accordance with the methods disclosed herein.

Binding small-molecule antagonists of the present disclosure may beidentified and chemically synthesized using known methodology (see,e.g., PCT Publication Nos. WO 00/00823 and WO 00/39585). In general,small molecule antagonists of the disclosure are usually less than about2000 daltons in size, alternatively less than about 1500, 750, 500, 250or 200 daltons in size, wherein such organic small molecules that arecapable of binding, preferably specifically, to a polypeptide asdescribed herein. These small molecule antagonists may be identifiedwithout undue experimentation using well-known techniques. In thisregard, it is noted that techniques for screening organic small-moleculelibraries for molecules that are capable of binding to a polypeptidetarget are well known in the art (see, e.g., international patentpublication Nos. WO00/00823 and WO00/39585).

Binding organic small molecules of the present disclosure may be, forexample, aldehydes, ketones, oximes, hydrazones, semicarbazones,carbazides, primary amines, secondary amines, tertiary amines,N-substituted hydrazines, hydrazides, alcohols, ethers, thiols,thioethers, disulfides, carboxylic acids, esters, amides, ureas,carbamates, carbonates, ketals, thioketals, acetals, thioacetals, arylhalides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromaticcompounds, heterocyclic compounds, anilines, alkenes, alkynes, diols,amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines,enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonylchlorides, diazo compounds, and acid chlorides.

E. Polynucleotide Antagonists

In other aspects, an ALK7:ActRIIB antagonist is a polynucleotide(ALK7:ActRIIB polynucleotide antagonist), or combination ofpolynucleotides. An ALK7:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, may inhibit, for example, oneor more ALK7:ActRIIB-binding ligands, type I receptors (e.g., ALK7),type II receptors (e.g., ActRIIB), co-receptor (e.g., Cripto orCryptic), and/or downstream signaling component (e.g., Smads). In someembodiments, ALK7:ActRIIB polynucleotide antagonist, or combination ofpolynucleotide antagonists, inhibits signaling mediated by one or moreALK7:ActRIIB-binding ligands, for example, as determined in a cell-basedassay such as those described herein. As described herein, ALK7:ActRIIBpolynucleotide antagonists may be used, alone or in combination with oneor more supportive therapies or active agents, to treat a patient inneed thereof (e.g., patients having kidney disease and/or a metabolicdisorder).

In some embodiments, an ALK7:ActRIIB polynucleotide antagonists, orcombination of polynucleotide antagonists, inhibits at least GDF11. Insome embodiments, an ALK7:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, inhibits at least GDF8. Insome embodiments, an ALK7:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, inhibits at least activin(activin A, activin B, activin C, activin E, activin AB, activin AC,activin AE, activin BC and/or activin BE). In some embodiments, anALK7:ActRIIB polynucleotide antagonist, or combination of polynucleotideantagonists, inhibits at least GDF11, GDF8, and activin. In someembodiments, an ALK7:ActRIIB polynucleotide antagonist, or combinationof polynucleotide antagonists, inhibits at least ALK7. In someembodiments, an ALK7:ActRIIB polynucleotide antagonist, or combinationof polynucleotide antagonists, inhibits at least ActRIIB In someembodiments, an ALK7:ActRIIB polynucleotide antagonist, or combinationof polynucleotide antagonists, inhibits at least Crypto or Cryptic. Insome embodiments, an ALK7:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, inhibits at least ActRIIB Insome embodiments, an ALK7:ActIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, inhibits at least BMP6. Insome embodiments, an ALK7:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, inhibits at least GDF3. Insome embodiments, an ALK7:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, inhibits at least BMP10. Insome embodiments, an ALK7:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, inhibits at least BMP5. Insome embodiments, an ALK7:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonist, inhibits at least nodal. Insome embodiments, an ALK7:ActRIIB polynucleotide antagonist, orcombination of polynucleotide antagonists, as disclosed herein does notinhibit or does not substantially inhibit BMP9.

In some embodiments, the polynucleotide antagonists of the disclosuremay be an antisense nucleic acid, an RNAi molecule [e.g., smallinterfering RNA (siRNA), small-hairpin RNA (shRNA), microRNA (miRNA)],an aptamer and/or a ribozyme. The nucleic acid and amino acid sequencesof human GDF11, GDF8, activin (activin A, activin B, activin C, andactivin E), BMP6, GDF3, BMP5, ALK7, ActRIIB, Cripto, Cryptic, Nodal, andBMP10 are known in the art. In addition, many different methods ofgenerating polynucleotide antagonists are well known in the art.Therefore polynucleotide antagonists for use in accordance with thisdisclosure may be routinely made by the skilled person in the art basedon the knowledge in the art and teachings provided herein.

Antisense technology can be used to control gene expression throughantisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed, for example, in Okano (1991) J. Neurochem.56:560; Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple-helix formationis discussed in, for instance, Cooney et al. (1988) Science 241:456; andDervan et al., (1991) Science 251:1300. The methods are based on bindingof a polynucleotide to a complementary DNA or RNA. In some embodiments,the antisense nucleic acids comprise a single-stranded RNA or DNAsequence that is complementary to at least a portion of an RNAtranscript of a gene disclosed herein. However, absolutecomplementarity, although preferred, is not required.

A sequence “complementary to at least a portion of an RNA,” referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids of a gene disclosed herein, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acid.Generally, the larger the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Polynucleotides that are complementary to the 5′ end of the message, forexample, the 5′-untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′-untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well [see, e.g., Wagner, R., (1994) Nature372:333-335]. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of a gene of the disclosure, couldbe used in an antisense approach to inhibit translation of an endogenousmRNA. Polynucleotides complementary to the 5′-untranslated region of themRNA should include the complement of the AUG start codon. Antisensepolynucleotides complementary to mRNA coding regions are less efficientinhibitors of translation but could be used in accordance with themethods of the present disclosure. Whether designed to hybridize to the5′-, 3′- or coding region of an mRNA of the disclosure, antisensenucleic acids should be at least six nucleotides in length, and arepreferably oligonucleotides ranging from 6 to about 50 nucleotides inlength. In specific aspects the oligonucleotide is at least 10nucleotides, at least 17 nucleotides, at least 25 nucleotides or atleast 50 nucleotides.

In one embodiment, the antisense nucleic acid of the present disclosureis produced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof is transcribed, producing anantisense nucleic acid (RNA) of a gene of the disclosure. Such a vectorwould contain a sequence encoding the desired antisense nucleic acid.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding desired genes of the instantdisclosure, or fragments thereof, can be by any promoter known in theart to act in vertebrate, preferably human cells. Such promoters can beinducible or constitutive. Such promoters include, but are not limitedto, the SV40 early promoter region [see, e.g., Benoist and Chambon(1981) Nature 290:304-310], the promoter contained in the 3′long-terminal repeat of Rous sarcoma virus [see, e.g., Yamamoto et al.(1980) Cell 22:787-797], the herpes thymidine promoter [see, e.g.,Wagner et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445], andthe regulatory sequences of the metallothionein gene [see, e.g.,Brinster, et al. (1982) Nature 296:39-42].

In some embodiments, the polynucleotide antagonists are interfering RNA(RNAi) molecules that target the expression of one or more of: GDF11,GDF8, activin (activin A, activin B, activin C, and activin E), BMP6,GDF3, BMP5, ALK7, ActRIIB, Cripto, Cryptic, nodal, and BMP10. RNAirefers to the expression of an RNA which interferes with the expressionof the targeted mRNA. Specifically, RNAi silences a targeted gene viainteracting with the specific mRNA through a siRNA (small interferingRNA). The ds RNA complex is then targeted for degradation by the cell.An siRNA molecule is a double-stranded RNA duplex of 10 to 50nucleotides in length, which interferes with the expression of a targetgene which is sufficiently complementary (e.g. at least 80% identity tothe gene). In some embodiments, the siRNA molecule comprises anucleotide sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100%identical to the nucleotide sequence of the target gene.

Additional RNAi molecules include short-hairpin RNA (shRNA); alsoshort-interfering hairpin and microRNA (miRNA). The shRNA moleculecontains sense and antisense sequences from a target gene connected by aloop. The shRNA is transported from the nucleus into the cytoplasm, andit is degraded along with the mRNA. Pol III or U6 promoters can be usedto express RNAs for RNAi. Paddison et al. [Genes & Dev. (2002)16:948-958, 2002] have used small RNA molecules folded into hairpins asa means to affect RNAi. Accordingly, such short-hairpin RNA (shRNA)molecules are also advantageously used in the methods described herein.The length of the stem and loop of functional shRNAs varies; stemlengths can range anywhere from about 25 to about 30 nt, and loop sizecan range between 4 to about 25 nt without affecting silencing activity.While not wishing to be bound by any particular theory, it is believedthat these shRNAs resemble the double-stranded RNA (dsRNA) products ofthe DICER RNase and, in any event, have the same capacity for inhibitingexpression of a specific gene. The shRNA can be expressed from alentiviral vector. An miRNA is a single-stranded RNA of about 10 to 70nucleotides in length that are initially transcribed as pre-miRNAcharacterized by a “stem-loop” structure, which are subsequentlyprocessed into mature miRNA after further processing through the RISC.

Molecules that mediate RNAi, including without limitation siRNA, can beproduced in vitro by chemical synthesis (Hohjoh, FEBS Lett 521:195-199,2002), hydrolysis of dsRNA (Yang et al., Proc Natl Acad Sci USA99:9942-9947, 2002), by in vitro transcription with T7 RNA polymerase(Donzeet et al., Nucleic Acids Res 30:e46, 2002; Yu et al., Proc NatlAcad Sci USA 99:6047-6052, 2002), and by hydrolysis of double-strandedRNA using a nuclease such as E. coli RNase III (Yang et al., Proc NatlAcad Sci USA 99:9942-9947, 2002).

According to another aspect, the disclosure provides polynucleotideantagonists including but not limited to, a decoy DNA, a double-strandedDNA, a single-stranded DNA, a complexed DNA, an encapsulated DNA, aviral DNA, a plasmid DNA, a naked RNA, an encapsulated RNA, a viral RNA,a double-stranded RNA, a molecule capable of generating RNAinterference, or combinations thereof.

In some embodiments, the polynucleotide antagonists of the disclosureare aptamers. Aptamers are nucleic acid molecules, includingdouble-stranded DNA and single-stranded RNA molecules, which bind to andform tertiary structures that specifically bind to a target molecule.The generation and therapeutic use of aptamers are well established inthe art (see, e.g., U.S. Pat. No. 5,475,096). Additional information onaptamers can be found in U.S. Patent Application Publication No.20060148748. Nucleic acid aptamers are selected using methods known inthe art, for example via the Systematic Evolution of Ligands byExponential Enrichment (SELEX) process. SELEX is a method for the invitro evolution of nucleic acid molecules with highly specific bindingto target molecules as described in, e.g., U.S. Pat. Nos. 5,475,096;5,580,737; 5,567,588; 5,707,796; 5,763,177; 6,011,577; and 6,699,843.Another screening method to identify aptamers is described in U.S. Pat.No. 5,270,163. The SELEX process is based on the capacity of nucleicacids for forming a variety of two- and three-dimensional structures, aswell as the chemical versatility available within the nucleotidemonomers to act as ligands (form specific binding pairs) with virtuallyany chemical compound, whether monomeric or polymeric, including othernucleic acid molecules and polypeptides. Molecules of any size orcomposition can serve as targets. The SELEX method involves selectionfrom a mixture of candidate oligonucleotides and step-wise iterations ofbinding, partitioning and amplification, using the same generalselection scheme, to achieve desired binding affinity and selectivity.Starting from a mixture of nucleic acids, which can comprise a segmentof randomized sequence, the SELEX method includes steps of contactingthe mixture with the target under conditions favorable for binding;partitioning unbound nucleic acids from those nucleic acids which havebound specifically to target molecules; dissociating the nucleicacid-target complexes; amplifying the nucleic acids dissociated from thenucleic acid-target complexes to yield a ligand enriched mixture ofnucleic acids. The steps of binding, partitioning, dissociating andamplifying are repeated through as many cycles as desired to yieldnucleic acid ligands which bind with high affinity and specificity tothe target molecule.

Typically, such binding molecules are separately administered to theanimal [see, e.g., O'Connor (1991) J. Neurochem. 56:560], but suchbinding molecules can also be expressed in vivo from polynucleotidestaken up by a host cell and expressed in vivo [see, e.g.,Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)].

F. Follistatin and FLRG Antagonists

It is known that members of the follistatin and FLRG group of proteinsantagonize ligands that signal through the ALK7:ActRIIB pathway.Accordingly, in other aspects, an ALK7:ActRIIB antagonist is afollistatin or FLRG polypeptide, which may be used alone or incombination with one or more additional supportive therapies and/oractive agents as disclosed herein to achieve a desired effect (e.g.,treat patients having kidney disease and/or a metabolic disorder).

The term “follistatin polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of follistatin as well as any variantsthereof (including mutants, fragments, fusions, and peptidomimeticforms) that retain a useful activity, and further includes anyfunctional monomer or multimer of follistatin. In certain preferredembodiments, follistatin polypeptides of the disclosure bind to and/orinhibit activin and/or GDF8 activity. Variants of follistatinpolypeptides that retain activin binding properties can be identifiedbased on previous studies involving follistatin and activininteractions. For example, WO2008/030367 discloses specific follistatindomains (“FSDs”) that are shown to be important for activin binding. Asshown below in SEQ ID NOs: 90-94, the follistatin N-terminal domain(“FSND” SEQ ID NO: 92), FSD2 (SEQ ID NO: 94), and to a lesser extentFSD1 (SEQ ID NO: 93) represent exemplary domains within follistatin thatare important for activin binding. In addition, methods for making andtesting libraries of polypeptides are described above in the context ofActRII polypeptides, and such methods also pertain to making and testingvariants of follistatin. Follistatin polypeptides include polypeptidesderived from the sequence of any known follistatin having a sequence atleast about 80% identical to the sequence of a follistatin polypeptide,and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greateridentity. Examples of follistatin polypeptides include the maturefollistatin polypeptide or shorter isoforms or other variants of thehuman follistatin precursor polypeptide (SEQ ID NO: 90) as described,for example, in WO2005/025601.

The human follistatin precursor polypeptide isoform FST344 is asfollows:

(SEQ ID NO: 90; NCBI Reference No. NP_037541.1)   1MVRARHQPGG LCLLLLLLCQ FMEDRSAQAG NCWLRQAKNG RCQVLYKTEL  51SKEECCSTGR LSTSWTEEDV NDNTLFKWMI FNGGAPNCIP CKETCENVDC 101GPGKKCRMNK KNKPRCVCAP DCSNITWKGP VCGLDGKTYR NECALLKARC 151KEQPELEVQY QGRCKKTCRD VFCPGSSTCV VDQTNNAYCV TCNRICPEPA 201SSEQYLCGND GVTYSSACHL RKATCLLGRS IGLAYEGKCI KAKSCEDIQC 251TGGKKCLWDF KVGRGRCSLC DELCPDSKSD EPVCASDNAT YASECAMKEA 301ACSSGVLLEV KHSGSCNSIS EDTEEEEEDE DQDYSFPISS ILEW

The signal peptide is underlined; also underlined above are the last 27residues which represent the C-terminal extension distinguishing thisfollistatin isoform from the shorter follistatin isoform FST317 shownbelow.

The human follistatin precursor polypeptide isoform FST317 is asfollows:

(SEQ ID NO: 91; NCBI Reference No. NP_006341.1)   1MVRARHQPGG LCLLLLLLCQ FMEDRSAQAG NCWLRQAKNG RCQVLYKTEL  51SKEECCSTGR LSTSWTEEDV NDNTLFKWMI FNGGAPNCIP CKETCENVDC 101GPGKKCRMNK KNKPRCVCAP DCSNITWKGP VCGLDGKTYR NECALLKARC 151KEQPELEVQY QGRCKKTCRD VFCPGSSTCV VDQTNNAYCV TCNRICPEPA 201SSEQYLCGND GVTYSSACHL RKATCLLGRS IGLAYEGKCI KAKSCEDIQC 251TGGKKCLWDF KVGRGRCSLC DELCPDSKSD EPVCASDNAT YASECAMKEA 301ACSSGVLLEV KHSGSCNThe signal peptide is underlined.

The follistatin N-terminal domain (FSND) sequence is as follows:

(SEQ ID NO: 92; FSND) GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCK

The FSD1 and FSD2 sequences are as follows:

(SEQ ID NO: 93; FSD1) ETCENVDCGPGKKCRWINKKNKPRCV (SEQ ID NO: 94; FSD2)KTCRDVFCPGSSTCVVDQTNNAYCVT

In other aspects, an ALK7:ActRIIB antagonist is a follistatin-likerelated gene (FLRG), also known as follistatin-related protein 3(FSTL3). The term “FLRG polypeptide” includes polypeptides comprisingany naturally occurring polypeptide of FLRG as well as any variantsthereof (including mutants, fragments, fusions, and peptidomimeticforms) that retain a useful activity. In certain embodiments, FLRGpolypeptides of the disclosure bind to and/or inhibit activin activity,particularly activin A. Variants of FLRG polypeptides that retainactivin binding properties can be identified using routine methods toassay FLRG and activin interactions (see, e.g., U.S. Pat. No.6,537,966). In addition, methods for making and testing libraries ofpolypeptides are described above in the context of ActRII and ALK7polypeptides and such methods also pertain to making and testingvariants of FLRG. FLRG polypeptides include polypeptides derived fromthe sequence of any known FLRG having a sequence at least about 80%identical to the sequence of an FLRG polypeptide, and optionally atleast 85%, 90%, 95%, 97%, 99% or greater identity.

The human FLRG precursor (follistatin-related protein 3 precursor)polypeptide is as follows:

1 MRPGAPGPLW PLPWGALAWA VGFVSSMGSG NPAPGGVCWL QQGQEATCSL  51VLQTDVTRAE CCASGNIDTA WSNLTHPGNK INLLGFLGLV HCLPCKDSCD  101GVECGPGKAC RMLGGRPRCE CAPDCSGLPA RLQVCGSDGA TYRDECELRA  151ARCRGHPDLS VMYRGRCRKS CEHVVCPRPQ SCVVDQTGSA HCVVCRAAPC  201PVPSSPGQEL CGNNNVTYIS SCHMRQATCF LGRSIGVRHA GSCAGTPEEP  251PGGESAEEEE NFV (SEQ ID NO: 95; NCBI Reference No. NP_005851.1) The signal peptide is underlined.

In certain embodiments, functional variants or modified forms of thefollistatin polypeptides and FLRG polypeptides include fusion proteinshaving at least a portion of the follistatin polypeptide or FLRGpolypeptide and one or more fusion domains, such as, for example,domains that facilitate isolation, detection, stabilization ormultimerization of the polypeptide. Suitable fusion domains arediscussed in detail above with reference to the ActRII polypeptides. Insome embodiment, an antagonist agent of the disclosure is a fusionprotein comprising an activin-binding portion of a follistatinpolypeptide fused to an Fc domain. In another embodiment, an antagonistagent of the disclosure is a fusion protein comprising an activinbinding portion of an FLRG polypeptide fused to an Fc domain.

G. Lefty A and B

The Lefty A and B proteins are known to regulate Nodal and otherproteins that signal through the ALK7:ActRIIB pathway. Accordingly, inother aspects, an ALK7:ActRIIB antagonist is a Lefty A or Lefty Bpolypeptide, which may be used alone or in combination with one or moreadditional supportive therapies and/or active agents as disclosed hereinto achieve a desired effect (e.g., treat kidney disease and/or ametabolic condition or disorder).

The term “Lefty A polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of Lefty A as well as any variantsthereof (including mutants, fragments, fusions, and peptidomimeticforms) that retain a useful activity, and further includes anyfunctional monomer or multimer of Lefty A. In certain preferredembodiments, Lefty A polypeptides of the disclosure binds to and/orinhibit nodal activity. In addition, methods for making and testinglibraries of polypeptides are described above in the context of ActRIIand ALK7 polypeptides, and such methods also pertain to making andtesting variants of Lefty A. Lefty A polypeptides include polypeptidesderived from the sequence of any known Lefty A having a sequence atleast about 80% identical to the sequence of a Lefty A polypeptide, andoptionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greateridentity. Examples of Lefty A polypeptides include the mature Lefty Apolypeptide or shorter isoforms or other variants of the human Lefty Aprecursor polypeptide (SEQ ID NO: 96).

The human Lefty A precursor polypeptide is as follows:

1 MWPLWLCWAL WVLPLAGPGA ALTEEQLLGS LLRQLQLSEV PVLDRADMEK LVIPAHVRAQ  61YVVLLRRSHG DRSRGKRFSQ SFREVAGRFL ASEASTHLLV FGMEQRLPPN SELVQAVLRL  121FQEPVPKAAL HRHGRLSPRS AQARVTVEWL RVRDDGSNRT SLIDSRLVSV HESGWKAFDV  181TEAVNFWQQL SRPRQPLLLQ VSVQREHLGP LASGAHKLVR FASQGAPAGL GEPQLELHTL  241DLRDYGAQGD CDPEAPMTEG TRCCRQEMYI DLQGMKWAKN WVLEPPGFLA YECVGTCQQP  301PEALAFNWPF LGPRQCIASE TASLPMIVSI KEGGRTRPQV VSLPNMRVQK CSCASDGALV  361PRRLQP (SEQ ID NO: 96; GenBank Id: AAD48145.1) The signal peptide is underlined.

The term “Lefty B polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of Lefty B as well as any variantsthereof (including mutants, fragments, fusions, and peptidomimeticforms) that retain a useful activity, and further includes anyfunctional monomer or multimer of Lefty B. In certain preferredembodiments, Lefty B polypeptides of the disclosure inhibit nodalactivity. In addition, methods for making and testing libraries ofpolypeptides are described above in the context of ActRII and ALK7polypeptides, and such methods also pertain to making and testingvariants of Lefty B. Lefty B polypeptides include polypeptides derivedfrom the sequence of any known Lefty B having a sequence at least about80% identical to the sequence of a Lefty B polypeptide, and optionallyat least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity. Examplesof Lefty B polypeptides include the mature Lefty B polypeptide orshorter isoforms or other variants of the human Lefty B precursorpolypeptide (SEQ ID NO: 97).

The human Lefty B precursor polypeptide is as follows:

1 MQPLWLCWAL WVLPLASPGA ALTGEQLLGS LLRQLQLKEV PTLDRADMEE LVIPTHVRAQ  61YVALLQRSHG DRSRGKRFSQ SFREVAGRFL ALEASTHLLV FGMEQRLPPN SELVQAVLRL  121FQEPVPKAAL HRHGRLSPRS ARARVTVEWL RVRDDGSNRT SLIDSRLVSV HESGWKAFDV  181TEAVNFWQQL SRPRQPLLLQ VSVQREHLGP LASGAHKLVR FASQGAPAGL GEPQLELHTL  241DLGDYGAQGD CDPEAPMTEG TRCCRQEMYI DLQGMKWAEN WVLEPPGFLA YECVGTCRQP  301PEALAFKWPF LGPRQCIASE TDSLPMIVSI KEGGRTRPQV VSLPNMRVQK CSCASDGALV  361PRRLQP (SEQ ID NO: 97; GenBank Id: AAD48144.1) The signal peptide is underlined.

In certain embodiments, functional variants or modified forms of theLefty A polypeptides and Lefty B polypeptides include fusion proteinshaving at least a portion of the Lefty A polypeptide or Leftypolypeptide and one or more fusion domains, such as, for example,domains that facilitate isolation, detection, stabilization ormultimerization of the polypeptide. Suitable fusion domains arediscussed in detail above with reference to the ActRII and ALK7polypeptides. In some embodiment, an antagonist agent of the disclosureis a fusion protein comprising a nodal-binding portion of a Lefty Aand/or Lefty B polypeptide fused to an Fc domain.

H. DAN-Related Proteins

Members of the DAN family of proteins are known to regulate ligands thatsignal through the ALK7:ActRIIB pathway. Accordingly, in other aspects,an ALK7:ActRIIB antagonist is a DAN-related protein (e.g., Cerberus andCoco), which may be used alone or in combination with one or moreadditional supportive therapies and/or active agents as disclosed hereinto achieve a desired effect (e.g., treat patients having kidney diseaseand/or a metabolic disorder).

The term “Cerberus polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of Cerberus as well as any variantsthereof (including mutants, fragments, fusions, and peptidomimeticforms) that retain a useful activity, and further includes anyfunctional monomer or multimer of Cerberus. In certain preferredembodiments, Cerberus polypeptides of the disclosure bind to and/orinhibit nodal activity. In addition, methods for making and testinglibraries of polypeptides are described above in the context of ActRIIand ALK7 polypeptides, and such methods also pertain to making andtesting variants of Cerberus. Cerberus polypeptides include polypeptidesderived from the sequence of any known Cerberus having a sequence atleast about 80% identical to the sequence of a Cerberus polypeptide, andoptionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greateridentity. Examples of Cerberus polypeptides include the mature Cerberuspolypeptide or shorter isoforms or other variants of the human Cerberusprecursor polypeptide (SEQ ID NO: 98).

The human Cerberus precursor polypeptide is as follows:

1 MHLLLFQLLV LLPLGKTTRH QDGRQNQSSL SPVLLPRNQR ELPTGNHEEA EEKPDLFVAV  61PHLVATSPAG EGQRQREKML SRFGRFWKKP EREMHPSRDS DSEPFPPGTQ SLIQPIDGMK  121MEKSPLREEA KKEWHHEMER KTPASQGVIL PIKSHEVHWE TCRTVPFSQT ITHEGCEKVV  181VQNNLCFGKC GSVHFPGAAQ HSHTSCSHCL PAKFTTMHLP LNCTELSSVI KVVMLVEECQ  241CKVKTEHEDG HILHAGSQDS FIPGVSA (SEQ IDNO: 98; NCBI Reference: NP_005445.1) The signal peptide is underlined.

The term “Coco polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of Coco as well as any variants thereof(including mutants, fragments, fusions, and peptidomimetic forms) thatretain a useful activity, and further includes any functional monomer ormultimer of Coco. In certain preferred embodiments, Coco polypeptides ofthe disclosure bind to and/or inhibit nodal activity. In addition,methods for making and testing libraries of polypeptides are describedabove in the context of ActRII and ALK7 polypeptides, and such methodsalso pertain to making and testing variants of Coco. Coco polypeptidesinclude polypeptides derived from the sequence of any known Coco havinga sequence at least about 80% identical to the sequence of a Cocopolypeptide, and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or greater identity. Examples of Coco polypeptides include the matureCoco polypeptide or shorter isoforms or other variants of the human Cocoprecursor polypeptide (SEQ ID NO: 99).

The human Coco precursor polypeptide is as follows:

1 MLLGQLSTLL CLLSGALPTG SGRPEPQSPR PQSWAAANQT WALGPGALPP LVPASALGSW  61KAFLGLQKAR QLGMGRLQRG QDEVAAVTLP LNPQEVIQGM CKAVPFVQVF SRPGCSAIRL  121RNHLCFGHCS SLYIPGSDPT PLVLCNSCMP ARKRWAPVVL WCLTGSSASR RRVKISTMLI  181EGCHCSPKA (SEQ ID NO: 99; GenBank Id:) The signal peptide is underlined.

In certain embodiments, functional variants or modified forms of theCerberus polypeptides and Coco polypeptides include fusion proteinshaving at least a portion of the Cerberus polypeptide and/or Cocopolypeptide and one or more fusion domains, such as, for example,domains that facilitate isolation, detection, stabilization ormultimerization of the polypeptide. Suitable fusion domains arediscussed in detail above with reference to the ActRII and ALK7polypeptides. In some embodiment, an antagonist agent of the disclosureis a fusion protein comprising a nodal-binding portion of a Cerberusand/or Coco polypeptide fused to an Fc domain.

3. Screening Assays

In certain embodiments, ALK7:ActRIIB receptor heteromultimers (e.g.,ALK7:ActRIIB heterodimers) may be used to generate and/or screen forALK7:ActRIIB inhibitors, particularly inhibitors that interfere withALK7-ligand and/or ALK7-Type II receptor (e.g., ActRIIB) interaction(e.g., anti-ALK7 antibodies).

As discussed herein, TGF-beta superfamily ligand signals are mediated bycomplexes of Type I and Type II kinase receptors, which phosphorylateand activate downstream Smad proteins upon ligand stimulation [see,e.g., Massague (2000) Nat. Rev. Mol. Cell. Biol. 1: 169-178]. Whileessential for signaling, Type I receptors generally have very weakaffinity for TGF-beta superfamily ligands. Instead, Type II receptorsgenerally bind to ligands with high affinity. This ligand bindingpromotes stable complex formation between Type I and II receptors,resulting in phosphorylation of type I receptors by Type II receptorsand thus activation of Smad proteins. In view of the weaker affinity, itis generally difficult to observe ligand binding activity of Type Ireceptors in isolation.

Indeed, the examples herein demonstrate that an ActRIIB homodimer bindsto many different ligands, many with high affinity. In contrast, an ALK7homodimer did not display affinity for any of the ligands examined. Asligands do not detectably bind to ALK7, it is difficult to screenfor/identify agents that can inhibit ALK7 activity. However, asdemonstrated by the examples herein, the ligand-binding activity of ALK7becomes measureable when paired with a type II receptor. Thus,ALK7:ActRIIB heteromultimers (e.g., heterodimers) are a useful tool foridentifying ALK7 inhibitors using various screening assays such asdescribed herein as well as those known in the art. Therefore, in someembodiments, the present disclosure relate to the use of ALK7:Type IIreceptor heteromultimers (e.g., ALK7:ActRIIB heteromultimers) toidentify (screen for) ALK7 inhibitors.

For example, ALK7:Type II receptor heteromultimers (e.g., ALK7:ActRIIBheteromultimers) may be particular useful for generating/identifyingantibodies that bind to ALK7, particularly antibodies that bind to andinhibit ALK7. While ALK7 polypeptides, including ALK7 homodimers asdescribed herein, may be used to generate/identify antibodies that bindto ALK7, simple binding is not sufficient to demonstrate that a givenALK7 antibody can inhibit an ALK7 activity (e.g., interfere withALK7-ligand or ALK7-Type II receptor interactions). As demonstratedherein, ALK7 polypeptides do not display ligand-binding activity in theabsence of a Type II receptor, making it difficult to identifyantibodies that can inhibit ALK7. ALK7:Type II receptor heteromultimers(e.g., ALK7:ActRIIB heteromultimers) solve this problem by providing afunctional read out for ALK7 activity. Moreover, without wishing to bebound to any particular mechanisms of action, association with a Type IIreceptor (e.g., ActRIIB) may improve the ability to generate antibodiesthat inhibit ALK7 activity. For example, association with a Type IIreceptor may help to stabilize ALK7 in such a way as to optimize theprocessing and presentation of important ALK7 epitopes that mediateligand-binding activities and thus improve the development of ALK7inhibitory antibodies. Therefore, in some embodiments, the presentdisclosure relates to the use of ALK7:Type II heteromultimers (e.g.,ALK7:ActRIIB heteromultimers) to generate antibodies that bind to andinhibit ALK7. Methods for generating and screening for antibodies arewell known in the art and described herein, and ALK7-Type IIheteromultimers (e.g., ALK7:ActRIIB heteromultimers) may be used inaccordance with one or more of these methods to generate/identifyantibodies that bind to and inhibit ALK7 activity.

In certain embodiments, ALK7:Type II receptor heterodimers (e.g.,ALK7:ActRIIB heteromultimers such as heterodimers) may be used toidentify activin C inhibitors, particularly inhibitors that bind toactivin C and interfere with activin C-Type I/II receptor interaction.

Surprisingly, the data presented herein demonstrates that ALK7:ActRIIBheterodimers bind to activin C. Insight into this novel interactionprovides an opportunity to develop/identify activin C inhibitors. Inaddition, given the structural similarity between activin C and activinE, it is expected that activin E also binds to ALK7:ActRIIB, and thusthis novel interaction may be used to identify activin E antagonists aswell. Heterodimeric ligands, such as activin AC, AE, BC and BE are alsoincluded in the reference to activin C or E, respectively in thissection of the disclosure. Agents that affect both activin C and E arealso contemplated. Various types of activin C or E antagonists areexpected to be useful in accordance with the methods described hereinincluding, for example, small molecules, antibodies, and inhibitornucleic acids, particularly those that disrupt activin C (orE)-ALK7:ActRIIB interaction. An agent (e.g., an antibody or smallmolecule) that is specifically reactive with activin C or E and whicheither binds to activin C or E so as to compete with its binding to ALK7and/or ActRIIB or otherwise inhibits activin C or E-mediated signalingmay be used as an antagonist of activin C or E. Likewise, an agent thatis specifically reactive with ALK7 and/or ActRIIB and which disruptsactivin C or E binding may be used as an antagonist. In regard toantibodies, immunogens can be derived from an activin C or Epolypeptide, ALK7 polypeptide, or ActRIIB polypeptide and used togenerate antibodies using standard methods known in the art. See, e.g.,Antibodies: A Laboratory Maneal ed. by Harlow and Lane, Cold SpringHarbor Press, 1988. Using various screening assays known in the art suchas those described herein, ALK7:Type II receptor heterodimers (e.g.,ALK7:ActRIIB heteromultimers such as heterodimers) may be used toidentify activin C or E inhibitors.

In certain aspects, the present disclosure relates to the use ofALK7:ActRIIB heteromultimer complexes to identify compounds (agents)which are agonists or antagonists of TGFβ superfamily receptors.Compounds identified through this screening can be tested to assesstheir ability to modulate tissue growth, such as bone, cartilage,muscle, fat, and/or neurons, growth in vivo or in vitro. These compoundscan be tested, for example, in animal models.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting TGFβ superfamily ligand signaling(e.g., SMAD signaling). In certain embodiments, high-throughputscreening of compounds can be carried out to identify agents thatperturb TGFβ superfamily receptor-mediated effects on a selected cellline. In certain embodiments, the assay is carried out to screen andidentify compounds that specifically inhibit or reduce binding of anALK7:ActRIIB heteromultimer to a binding partner (e.g., activin, GDF11,GDF8, BMP6, GDF3, BMP5, nodal, and BMP10). Alternatively, the assay canbe used to identify compounds that enhance binding of an ALK7:ActRIIBheteromultimer to a ligand. In a further embodiment, the compounds canbe identified by their ability to interact with an ALK7:ActRIIBheteromultimer.

In some embodiments, the present disclosure relates to the use of anALK7:ActRIIB heteromultimer and activin C or E polypeptides to identifycompounds which are agonists or antagonists of the activin C (orE)-ALK7:ActRIIB signaling pathway. Compounds identified through thisscreening assay can be tested to assess their ability to modulateactivin C or E signaling activity in vitro. Optionally, these compoundscan further be tested in animal models to assess their ability tomodulate tissue growth in vivo. There are numerous approaches toscreening for therapeutic agents for modulating tissue growth bytargeting activin C or E and ALK7:ActRIIB heteromultimers. In certainembodiments, high-throughput screening of compounds can be carried outto identify agents that perturb activin C or E or ALK7:ActRIIB-mediatedcell signaling. In certain embodiments, the assay is carried out toscreen and identify compounds that specifically inhibit or reducebinding of an ALK7:ActRIIB heteromultimer to activin C or E.Alternatively, the assay can be used to identify compounds that enhancebinding of an ALK7:ActRIIB heteromultimer to activin C or E. In afurther embodiment, the compounds can be identified by their ability tointeract with activin C or E or an ALK7:ActRIIB heteromultimer.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. Incertain embodiments, the test agent is a small organic molecule having amolecular weight of less than about 2,000 Daltons.

The test compounds of the disclosure can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S-transferase (GST),photoactivatable crosslinkers or any combinations thereof.

In many drug-screening programs which test libraries of compounds andnatural extracts, high-throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between anALK7:ActRIIB heteromultimer complex and a binding partner (e.g., activinC or E).

Merely to illustrate, in an exemplary screening assay of the presentdisclosure, the compound of interest is contacted with an isolated andpurified ALK7:ActRIIB heteromultimer which is ordinarily capable ofbinding to activin C, as appropriate for the intention of the assay. Tothe mixture of the compound and ALK7:ActRIIB heteromultimer complex isthen added to a composition containing activin C or E. Detection andquantification of ALK7:ActRIIB heteromultimer-activin C or E complexesprovides a means for determining the compound's efficacy at inhibiting(or potentiating) complex formation between the ALK7:ActRIIBheteromultimer and activin C or E. The efficacy of the compound can beassessed by generating dose-response curves from data obtained usingvarious concentrations of the test compound. Moreover, a control assaycan also be performed to provide a baseline for comparison. For example,in a control assay, isolated and purified activin C or E is added to acomposition containing the ALK7:ActRIIB heteromultimer, and theformation of ALK7:ActRIIB heteromultimer-activin C or E is quantitatedin the absence of the test compound. It will be understood that, ingeneral, the order in which the reactants may be admixed can be varied,and can be admixed simultaneously. Moreover, in place of purifiedproteins, cellular extracts and lysates may be used to render a suitablecell-free assay system.

Binding of a ALK7:ActRIIB heteromultimer complex to another protein(e.g., activin C or E) may be detected by a variety of techniques. Forinstance, modulation of the formation of complexes can be quantitatedusing, for example, detectably labeled proteins such as radiolabeled(e.g., ³²P, ³⁵S, ¹⁴C or ³H), fluorescently labeled (e.g., FITC), orenzymatically labeled ALK7:ActRIIB heteromeric complex and/or a bindingpartner (e.g., activin C or E), by immunoassay, or by chromatographicdetection.

In certain embodiments, the present disclosure contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between an ALK7:ActRIIB heteromultimer and abinding protein (e.g., activin C or E). Further, other modes ofdetection, such as those based on optical waveguides (PCT Publication WO96/26432 and U.S. Pat. No. 5,677,196), surface plasmon resonance (SPR),surface charge sensors, and surface force sensors, are compatible withmany embodiments of the disclosure.

Moreover, the present disclosure contemplates the use of an interactiontrap assay, also known as the “two-hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between an ALK7:ActRIIBheteromultimer and a binding partner (e.g., activin C or E). See, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene8:1693-1696). In a specific embodiment, the present disclosurecontemplates the use of reverse two-hybrid systems to identify compounds(e.g., small molecules or peptides) that dissociate interactions betweenan ALK7:ActRIIB heteromultimer and a binding protein (e.g., activin C orE) [Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29; Vidal andLegrain, (1999) Trends Biotechnol 17:374-81; and U.S. Pat. Nos.5,525,490; 5,955,280; and 5,965,368].

In certain embodiments, the subject compounds are identified by theirability to interact with an ALK7:ActRIIB heteromultimer or activin C orE of the disclosure. The interaction between the compound and anALK7:ActRIIB heteromultimer or activin C or E may be covalent ornon-covalent. For example, such interaction can be identified at theprotein level using in vitro biochemical methods, includingphoto-crosslinking, radiolabeled ligand binding, and affinitychromatography [Jakoby W B et al. (1974) Methods in Enzymology 46:1]. Incertain cases, the compounds may be screened in a mechanism-based assay,such as an assay to detect compounds which bind to an ALK7:ActRIIBheteromultimer or activin C or E. This may include a solid-phase orfluid-phase binding event. Alternatively, the gene encoding anALK7:ActRIIB heteromultimer or activin C or E can be transfected with areporter system (e.g., β-galactosidase, luciferase, or green fluorescentprotein) into a cell and screened against the library preferably byhigh-throughput screening or with individual members of the library.Other mechanism-based binding assays may be used; for example, bindingassays which detect changes in free energy. Binding assays can beperformed with the target fixed to a well, bead or chip or captured byan immobilized antibody or resolved by capillary electrophoresis. Thebound compounds may be detected usually using colorimetric endpoints orfluorescence or surface plasmon resonance.

4. Exemplary Therapeutic Uses

In certain embodiments, an ALK7:ActRIIB antagonist, or combinations ofsuch antagonists, of the present disclosure can be used to treat orprevent a disease or condition that is associated with abnormal activityof an ALK7:ActRIIB-binding ligand. These diseases, disorders, orconditions are generally referred to herein as “ALK7:ActRIIB-associatedconditions” or “ALK7:ActRIIB-associated disorders.” In certainembodiments, the present disclosure provides methods of treating orpreventing an ALK7:ActRIIB-associated condition in an individual byadministering to an individual in need thereof a therapeuticallyeffective amount of an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheteromultimer such as an ALK7:ActRIIB heterodimer), or combinations ofsuch antagonists, as described herein. The terms “subject,” an“individual,” or a “patient” are interchangeable throughout thespecification. Any of the ALK7:ActRIIB antagonists of the disclosure canpotentially be employed individually or in combination for therapeuticuses disclosed herein. These methods are particularly aimed attherapeutic and prophylactic treatments of mammals including, forexample, rodents, primates, and humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes amelioration or elimination of the condition once it has beenestablished. In either case, prevention or treatment may be discerned inthe diagnosis provided by a physician or other health care provider andthe intended result of administration of the therapeutic agent.

In general, treatment or prevention of a disease or condition asdescribed in the present disclosure is achieved by administering anALK7:ActRIIB antagonist, or combinations of such antagonists, of thepresent disclosure in an “effective amount”. An effective amount of anagent refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result. A“therapeutically effective amount” of an agent of the present disclosuremay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the agent to elicit adesired response in the individual. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result.

The kidneys maintain many features of the blood, including volume, pHbalance, electrolyte concentrations, and blood pressure, as well asbearing responsibility for toxin and waste filtration. These functionsdepend upon the intricate structure of the kidney nephrons, constantflow of blood through the various capillaries of the kidney, and theregulation of the kidney by signals from the rest of the body, includingendocrine hormones. Problems with kidney function manifest by directmechanisms (e.g. genetic defects, infection, or toxin exposure) and byindirect mechanisms progressively proceeding from long term stressorslike hypertrophy and hyperfiltration (themselves often a result of moredirect insults to kidney function). Due to the central role of thekidney in blood maintenance and waste secretion, kidney-associateddisease manifestations are many and varied; they can be reviewed inHarrison's Principles of Internal Medicine, 18^(th) edition, McGrawHill, N.Y., Part 13, Chp 277-289.

As described herein, an ALK7:ActRIIB antagonist had various beneficialeffects in a kidney disease model. In particular, treatment with anALK7:ActRIIB heteromultimer reduced kidney tissue damage, inflammation,and fibrosis in subjects having unilateral ureteral obstruction. Thesedata indicate that ALK7:ActRIIB antagonist may be used to treat orprevent kidney disease, particularly treating or preventing variouscomplications (manifestations) of kidney disease including, for example,kidney tissue damage, inflammation, and/or fibrosis.

Therefore, methods of this invention can be applied to variouskidney-associated diseases or conditions. As used herein,“kidney-associated disease or condition” can refer to any disease,disorder, or condition that affects the kidneys or the renal system.Examples of kidney-associated diseases or conditions include, but arenot limited to, chronic kidney diseases (or failure), acute kidneydiseases (or failure), primary kidney diseases, non-diabetic kidneydiseases, glomerulonephritis, interstitial nephritis, diabetic kidneydiseases, diabetic nephropathy, glomerulosclerosis, rapid progressiveglomerulonephritis, renal fibrosis, Alport syndrome, IDDM nephritis,mesangial proliferative glomerulonephritis, membranoproliferativeglomerulonephritis, crescentic glomerulonephritis, renal interstitialfibrosis, focal segmental glomerulosclerosis, membranous nephropathy,minimal change disease, pauci-immune rapid progressiveglomerulonephritis, IgA nephropathy, polycystic kidney disease, Dent'sdisease, nephrocytinosis, Heymann nephritis, autosomal dominant (adult)polycystic kidney disease, autosomal recessive (childhood) polycystickidney disease, acute kidney injury, nephrotic syndrome, renal ischemia,podocyte diseases or disorders, proteinuria, glomerular diseases,membranous glomerulonephritis, focal segmental glomerulonephritis,pre-eclampsia, eclampsia, kidney lesions, collagen vascular diseases,benign orthostatic (postural) proteinuria, IgM nephropathy, membranousnephropathy, sarcoidosis, diabetes mellitus, kidney damage due to drugs,Fabry's disease, aminoaciduria, Fanconi syndrome, hypertensivenephrosclerosis, interstitial nephritis, Sickle cell disease,hemoglobinuria, myoglobinuria, Wegener's Granulomatosis, GlycogenStorage Disease Type 1, chronic kidney disease, chronic renal failure,low Glomerular Filtration Rate (GFR), nephroangiosclerosis, lupusnephritis, ANCA-positive pauci-immune crescentic glomerulonephritis,chronic allograft nephropathy, nephrotoxicity, renal toxicity, kidneynecrosis, kidney damage, glomerular and tubular injury, kidneydysfunction, nephritic syndrome, acute renal failure, chronic renalfailure, proximal tubal dysfunction, acute kidney transplant rejection,chronic kidney transplant rejection, non-IgA mesangioproliferativeglomerulonephritis, postinfectious glomerulonephritis, vasculitides withrenal involvement of any kind, any hereditary renal disease, anyinterstitial nephritis, renal transplant failure, kidney cancer, kidneydisease associated with other conditions (e.g., hypertension, diabetes,and autoimmune disease), Dent's disease, nephrocytinosis, Heymannnephritis, a primary kidney disease, a collapsing glomerulopathy, adense deposit disease, a cryoglobulinemia-associated glomerulonephritis,an Henoch-Schonlein disease, a postinfectious glomerulonephritis, abacterial endocarditis, a microscopic polyangitis, a Churg-Strausssyndrome, an anti-GBM-antibody mediated glomerulonephritis, amyloidosis,a monoclonal immunoglobulin deposition disease, a fibrillaryglomerulonephritis, an immunotactoid glomerulopathy, ischemic tubularinjury, a medication-induced tubulo-interstitial nephritis, a toxictubulo-interstitial nephritis, an infectious tubulo-interstitialnephritis, a bacterial pyelonephritis, a viral infectioustubulo-interstitial nephritis which results from a polyomavirusinfection or an HIV infection, a metabolic-induced tubulo-interstitialdisease, a mixed connective disease, a cast nephropathy, a crystalnephropathy which may results from urate or oxalate or drug-inducedcrystal deposition, an acute cellular tubulo-interstitial allograftrejection, a tumoral infiltrative disease which results from a lymphomaor a post-transplant lymphoproliferative disease, an obstructive diseaseof the kidney, vascular disease, a thrombotic microangiopathy, anephroangiosclerosis, an atheroembolic disease, a mixed connectivetissue disease, a polyarteritis nodosa, a calcineurin-inhibitorinduced-vascular disease, an acute cellular vascular allograftrejection, an acute humoral allograft rejection, early renal functiondecline (ERFD), end stage renal disease (ESRD), renal vein thrombosis,acute tubular necrosis, acute interstitial nephritis, establishedchronic kidney disease, renal artery stenosis, ischemic nephropathy,uremia, drug and toxin-induced chronic tubulointerstitial nephritis,reflux nephropathy, kidney stones, Goodpasture's syndrome, normocyticnormochromic anemia, renal anemia, diabetic chronic kidney disease,IgG4-related disease, von Hippel-Lindau syndrome, tuberous sclerosis,nephronophthisis, medullary cystic kidney disease, renal cell carcinoma,adenocarcinoma, nephroblastoma, lymphoma, leukemia, hyposialylationdisorder, chronic cyclosporine nephropathy, renal reperfusion injury,renal dysplasia, azotemia, bilateral arterial occlusion, acute uric acidnephropathy, hypovolemia, acute bilateral obstructive uropathy,hypercalcemic nephropathy, hemolytic uremic syndrome, acute urinaryretention, malignant nephrosclerosis, postpartum glomerulosclerosis,scleroderma, non-Goodpasture's anti-GBM disease, microscopicpolyarteritis nodosa, allergic granulomatosis, acute radiationnephritis, post-streptococcal glomerulonephritis, Waldenstrom'smacroglobulinemia, analgesic nephropathy, arteriovenous fistula,arteriovenous graft, dialysis, ectopic kidney, medullary sponge kidney,renal osteodystrophy, solitary kidney, hydronephrosis, microalbuminuria,uremia, haematuria, hyperlipidemia, hypoalbuminaemia, lipiduria,acidosis, hyperkalemia, and edema.

In some embodiments, an ALK7:ActRIIB antagonist, or combinations of suchantagonists, of the present disclosure (e.g., ALK7:ActRIIBheteromultimers such as an ALK7:ActRIIB heterodimer) may be used totreat or prevent chronic kidney disease, optionally in combination withone or more supportive therapies for treating chronic kidney disease. Insome embodiments, an ALK7:ActRIIB antagonist, or combinations of suchantagonists, of the present disclosure (e.g., ALK7:ActRIIBheteromultimers such as an ALK7:ActRIIB heterodimer) may be used totreat or prevent one or more complications (symptoms or manifestations)of chronic kidney disease (e.g., tissue damage, inflammation, and/orfibrosis), optionally in combination with one or more supportivetherapies for treating chronic kidney disease. In some embodiments, anALK7:ActRIIB antagonist, or combinations of such antagonists, of thepresent disclosure (e.g., ALK7:ActRIIB heteromultimers such as anALK7:ActRIIB heterodimer) may be used to treat or prevent end-stagekidney failure, optionally in combination with one or more supportivetherapies for treating end-stage kidney disease. Chronic kidney disease(CKD), also known as chronic renal disease, is a progressive loss inrenal function over a period of months or years. The symptoms ofworsening kidney function may include feeling generally unwell andexperiencing a reduced appetite. Often, chronic kidney disease isdiagnosed as a result of screening of people known to be at risk ofkidney problems, such as those with high blood pressure or diabetes andthose with a blood relative with CKD. This disease may also beidentified when it leads to one of its recognized complications, such ascardiovascular disease, anemia, or pericarditis. Recent professionalguidelines classify the severity of CKD in five stages, with stage 1being the mildest and usually causing few symptoms and stage 5 being asevere illness with poor life expectancy if untreated. Stage 5 CKD isoften called end-stage kidney disease, end-stage renal disease, orend-stage kidney failure, and is largely synonymous with the nowoutdated terms chronic renal failure or chronic kidney failure; andusually means the patient requires renal replacement therapy, which mayinvolve a form of dialysis, but ideally constitutes a kidney transplant.CKD is initially without specific symptoms and is generally onlydetected as an increase in serum creatinine or protein in the urine. Asthe kidney function decreases, various symptoms may manifest asdescribed below. Blood pressure may be increased due to fluid overloadand production of vasoactive hormones created by the kidney via therenin-angiotensin system, increasing one's risk of developinghypertension and/or suffering from congestive heart failure. Urea mayaccumulate, leading to azotemia and ultimately uremia (symptoms rangingfrom lethargy to pericarditis and encephalopathy). Due to its highsystemic circulation, urea is excreted in eccrine sweat at highconcentrations and crystallizes on skin as the sweat evaporates (“uremicfrost”). Potassium may accumulate in the blood (hyperkalemia with arange of symptoms including malaise and potentially fatal cardiacarrhythmias). Hyperkalemia usually does not develop until the glomerularfiltration rate falls to less than 20-25 ml/min/1.73 m2, at which pointthe kidneys have decreased ability to excrete potassium. Hyperkalemia inCKD can be exacerbated by acidemia (which leads to extracellular shiftof potassium) and from lack of insulin. Erythropoietin synthesis may bedecreased causing anemia. Fluid volume overload symptoms may occur,ranging from mild edema to life-threatening pulmonary edema.Hyperphosphatemia, due to reduced phosphate excretion, may occurgenerally following the decrease in glomerular filtration.Hyperphosphatemia is associated with increased cardiovascular risk,being a direct stimulus to vascular calcification. Hypocalcemia maymanifest, which is generally caused by stimulation of fibroblast growthfactor-23. Osteocytes are responsible for the increased production ofFGF23, which is a potent inhibitor of the enzyme 1-alpha-hydroxylase(responsible for the conversion of 25-hydroxycholecalciferol into 1,25dihydroxyvitamin D3). Later, this progresses to secondaryhyperparathyroidism, renal osteodystrophy, and vascular calcificationthat further impairs cardiac function. Metabolic acidosis (due toaccumulation of sulfates, phosphates, uric acid etc.) may occur andcause altered enzyme activity by excess acid acting on enzymes; and alsoincreased excitability of cardiac and neuronal membranes by thepromotion of hyperkalemia due to excess acid (acidemia). Acidosis isalso due to decreased capacity to generate enough ammonia from the cellsof the proximal tubule. Iron deficiency anemia, which increases inprevalence as kidney function decreases, is especially prevalent inthose requiring haemodialysis. It is multifactoral in cause, butincludes increased inflammation, reduction in erythropoietin, andhyperuricemia leading to bone marrow suppression. People with CKD sufferfrom accelerated atherosclerosis and are more likely to developcardiovascular disease than the general population. Patients afflictedwith CKD and cardiovascular disease tend to have significantly worseprognoses than those suffering only from the latter.

In another embodiment, an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combinations of such antagonists, may be used inpatients with chronic kidney disease mineral bone disorder (CKD-MBD), abroad syndrome of interrelated skeletal, cardiovascular, andmineral-metabolic disorders arising from kidney disease. CKD-MBDencompasses various skeletal pathologies often referred to as renalosteodystrophy (ROD), which is a preferred embodiment for treatmentwith, an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIB heterodimer), orcombinations of such antagonists. Depending on the relative contributionof different pathogenic factors, ROD is manifested as diverse pathologicpatterns of bone remodeling (Hruska et al., 2008, Chronic kidney diseasemineral bone disorder (CKD-MBD); in Rosen et al. (ed) Primer on theMetabolic Bone Diseases and Disorders of Mineral Metabolism, 7th ed.American Society for Bone and Mineral Research, Washington D.C., pp343-349). At one end of the spectrum is ROD with uremic osteodystrophyand low bone turnover, characterized by a low number of activeremodeling sites, profoundly suppressed bone formation, and low boneresorption. At the other extreme is ROD with hyperparathyroidism, highbone turnover, and osteitis fibrosa. Given that an ALK7:ActRIIBantagonist (e.g., an ALK7:ActRIIB heterodimer), or combinations of suchantagonists, may exert both anabolic and antiresorptive effects, theseagents may be useful in patients across the ROD pathology spectrum.

In other embodiments, an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combinations of such antagonists, can be used forregulating body fat content in an animal and for treating or preventingconditions related thereto, and particularly, health-compromisingconditions related thereto. According to the present invention, toregulate (control) body weight can refer to reducing or increasing bodyweight, reducing or increasing the rate of weight gain, or increasing orreducing the rate of weight loss, and also includes activelymaintaining, or not significantly changing body weight (e.g., againstexternal or internal influences which may otherwise increase or decreasebody weight). One embodiment of the present disclosure relates toregulating body weight by administering to an animal (e.g., a human) inneed thereof an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combinations of such antagonists. For example, in someembodiments, an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combinations of such antagonists, may be used to treator prevent a disorder or condition selected from obesity (e.g.,abdominal obesity); overweight; insulin resistance; metabolic syndromeand other metabolic diseases or conditions; a lipid disorder such as,low HDL levels, high LDL levels, hyperlipidemia, hypertriglyceridemia ordyslipidemia; lipoprotein aberrations; decreased triglycerides;inflammation (e.g., liver inflammation and/or inflammation of adiposetissue), fatty liver disease; non-alcoholic fatty liver disease;hyperglycemia; impaired glucose tolerance (IGT); hyperinsulinemia; highcholesterol (e.g., high LDL levels and hypercholesterolemia);cardiovascular disease such as, heart disease including coronary heartdisease, congestive heart failure, stroke, peripheral vascular disease,atherosclerosis; arteriosclerosis, and hypertension; Syndrome X;vascular restenosis; neuropathy; retinopathy; neurodegenerative disease;endothelial dysfunction, respiratory dysfunction; pancreatitis;polycystic ovarian syndrome; elevated uric acid levels; haemochromatosis(iron overload); acanthosis nigricans (dark patches on the skin); orcancer (e.g., ovarian, breast, endometrial, and colon cancer); or aanother disorders/conditions associated with one or more of the abovediseases or conditions. In some embodiments, the disease or conditiontreated using an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combinations of such antagonists, is associated withoverweight (e.g., BMI of ≥25 kg/m²), or with too much body fat.

In one embodiment, the disclosure provides a method of reducing bodyweight comprising administering to a subject desiring to reduce bodyweight, or in need thereof, an effective amount of an ALK7:ActRIIBantagonist (e.g., an ALK7:ActRIIB heterodimer), or combinations of suchantagonists. In some embodiments, the subject is overweight (e.g.,pre-obese). In some embodiments, the subject has a body mass index (BMI)of 25 kg/m² or greater. In further embodiments, the subject has a BMI of25 kg/m² to 29.9 kg/m², 30 kg/m² to 39.9 kg/m², 25 kg/m² to 39.9 kg/m²,or 25 kg/m² to 50 kg/m². In some embodiments, the subject is obese. Insome embodiments, the subject has a BMI of 30 kg/m² or greater (e.g., 30to 39.9 kg/m² or 30 kg/m² to 50 kg/m²). In some embodiments, the subjectis morbidly obese. In some embodiments, the subject has a BMI of 40kg/m² or greater. In further embodiments, the subject has a BMI of 40kg/m² to 45 kg/m², or 40 kg/m² to 50 kg/m². In some embodiments, thesubject has central obesity (e.g., excess adiposity in the abdominalregion, including belly fat and/or visceral fat). In some embodiments,the subject has a waist/hip circumference ratio (WHR) of 0.85 orgreater. In some embodiments, the subject has peripheral obesity (e.g.,excess adiposity on the hips). In some embodiments, the subject has type2 diabetes mellitus. The ALK7:ActRIIB antagonist, or combination ofantagonists, may administered alone or as a combination therapy othertype of supportive therapy. For example, in some embodiments, thesupportive therapy is diet and/or exercise.

In one embodiment, the disclosure provides a method of reducing weightgain comprising administering to a subject desiring to reduce weightgain, or in need thereof, an effective amount of an ALK7:ActRIIBantagonist (e.g., an ALK7:ActRIIB heterodimer), or combination of suchantagonists. In some embodiments, the subject is overweight (e.g.,pre-obese). In some embodiments, the subject has a BMI of 25 kg/m² orgreater. In further embodiments, the subject has a BMI of 25 kg/m² to29.9 kg/m², 30 kg/m² to 39.9 kg/m², 25 kg/m² to 39.9 kg/m², or 25 kg/m²to 50 kg/m². In some embodiments, the subject is obese. In someembodiments, the subject has a BMI of 30 kg/m² or greater (e.g., 30 to39.9 kg/m² or 30 kg/m² to 50 kg/m²). In some embodiments, the subject ismorbidly obese. In some embodiments, the subject has a BMI of 40 kg/m²or greater. In further embodiments, the subject has a BMI of 40 kg/m² to45 kg/m², or 40 kg/m² to 50 kg/m². In some embodiments, the subject hastype 2 diabetes mellitus.

Also provided is a method of treating or preventing a disease orcondition associated with excess body weight, comprising administeringto a subject in need of treatment or prevention, an effective amount ofan ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIB heterodimer), orcombination of such antagonists. In one embodiment, the treated orprevented disease or condition is obesity. In one embodiment, thetreated or prevented disease or condition is insulin resistance. In oneembodiment, the treated or prevented disease or condition is a memberselected from the group consisting of: dyslipidemia, hyperlipidemia(total cholesterol level >240 mg/dL), hypercholesterolemia (e.g., totalcholesterol level of >200 mg/dL, >220 mg/dL, >240 mg/dL, >250 mg/dL,or >275 mg/dL), low HDL serum level (e.g., <40 mg/dL, <45 mg/dL, or <50mg/dL), high LDL serum level (e.g., ≥100 mg/dL, ≥130 mg/dL, ≥160 mg/dL,or ≥190 mg/dL), and hypertriglyceridemia (e.g., a fasting TG level of≥150 mg/dL, ≥175 mg/dL, ≥200 mg/dL, ≥300 mg/dL, ≥400 mg/dL, or ≥499mg/dL). In certain instances, the ALK7:ActRIIB antagonists treatment isan adjunct to diet and/or exercise.

In another embodiment the disclosure provides a method of reducing bodyweight in a subject who is overweight. The method includes administeringto an overweight subject an effective amount of an ALK7:ActRIIBantagonist (e.g., an ALK7:ActRIIB heterodimer), or combination of suchantagonists. In some embodiments, the subject has a body mass index(BMI) of 25 kg/m² or greater. In further embodiments, the subject has aBMI of 25 kg/m² to 29.9 kg/m², 30 kg/m² to 39.9 kg/m², 25 kg/m² to 39.9kg/m², or 25 kg/m² to 50 kg/m²′ or 27 to 40 kg/m². In some embodiments,the subject is obese. In some embodiments, the subject has a BMI of 30kg/m² or greater (e.g., 30 to 39.9 kg/m² or 30 kg/m² to 50 kg/m²). TheALK7-binding protein is administered alone or as a combination therapy.In some embodiments, the ALK7:ActRIIB antagonist treatment is an adjunctto diet and/or exercise.

In one embodiment the disclosure provides a method of reducing bodyweight in an obese subject. The method includes administering to thesubject an effective amount of an ALK7:ActRIIB antagonist (e.g., anALK7:ActRIIB heterodimer), or combination of such antagonists. In someembodiments, the subject has a BMI of 30 kg/m² or greater (e.g., 30 to39.9 kg/m² or 30 kg/m² to 50 kg/m². In some embodiments, the subject hasa BMI of 40 kg/m² or greater. In some embodiments, the subject hascentral obesity (e.g., excess adiposity in the abdominal region,including belly fat and/or visceral fat). In some embodiments, thesubject has a waist/hip circumference ratio (WHR) of 0.85 or greater. Insome embodiments, the subject has peripheral obesity (e.g., excessadiposity on the hips). In some embodiments, the ALK7:ActRIIB antagonisttreatment is an adjunct to diet and/or exercise.

In another embodiment, the disclosure provides a method of treatingand/or ameliorating obesity or a disease or condition associated withobesity, comprising administering to an obese subject, an effectiveamount of an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combination of such antagonists. In some embodiments,the subject has a BMI of 30 kg/m² or greater. In further embodiments,the subject has a BMI of 30 to 39.9 kg/m² or 30 kg/m² to 50 kg/m². Insome embodiments, the subject is morbidly obese. In some embodiments,the subject has a body BMI of 40 kg/m² or greater. In furtherembodiments, the subject has a BMI of 40 kg/m² to 45 kg/m², or 40 kg/m²to 50 kg/m²In some embodiments, the subject has type 2 diabetesmellitus. In some embodiments, the subject has a BMI of 30 kg/m² orgreater (e.g., 30 to 39.9 kg/m²). In some embodiments, the subject has aBMI of at least 40 kg/m². In some embodiments, the subject has centralobesity (e.g., excess adiposity in the abdominal region, including bellyfat and/or visceral fat). In some embodiments, the subject has awaist/hip circumference ratio (WHR) of 0.85 or greater. In someembodiments, the subject has peripheral obesity (e.g., excess adiposityon the hips). In some embodiments, the ALK7:ActRIIB antagonist treatmentis an adjunct to diet and/or exercise.

Also provided is a method of treating or preventing a disease orcondition associated with obesity, comprising administering to a subjectin need of treatment or prevention, an effective amount of anALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIB heterodimer), orcombination of such antagonists. In one embodiment, the treated orprevented disease or condition is a member selected from the groupconsisting of: dyslipidemia, hyperlipidemia (total cholesterollevel >240 mg/dL), hypercholesterolemia (e.g., total cholesterol levelof >200 mg/dL, >220 mg/dL, >240 mg/dL, >250 mg/dL, or >275 mg/dL), lowHDL serum level (e.g., <40 mg/dL, <45 mg/dL, or <50 mg/dL), high LDLserum level (e.g., ≥100 mg/dL, ≥130 mg/dL, ≥160 mg/dL, or ≥190 mg/dL),and hypertriglyceridemia (e.g., a fasting TG level of ≥150 mg/dL, ≥175mg/dL, ≥200 mg/dL, ≥300 mg/dL, ≥400 mg/dL, or ≥499 mg/dL). In oneembodiment, the treated or prevented disease or condition iscardiovascular disease. In an additional embodiment, the treated orprevented disease or condition is hypertension (high blood pressure),myocardial infarction, peripheral artery disease, vasoregulatoindysfunction, arteriosclerosis congestive heart failure, atherosclerosis,coronary heart disease, or microvascular disease. In one embodiment, thetreated or prevented disease or condition is liver disease. In oneembodiment, the treated or prevented liver disease or condition isNAFLD. In one embodiment, the liver disease is fatty liver. In oneembodiment, the liver disease is NASH. In another embodiment, thetreated or prevented disease or condition is a member selected from thegroup: steatohepatitis, steatosis, fibrosis, and/or cirrhosis. Incertain instances, the ALK7:ActRIIB antagonist treatment is an adjunctto diet and/or exercise.

In another embodiment, the disclosure provides a method of treating,ameliorating, and/or preventing type 2 diabetes mellitus or a disease orcondition associated with diabetes comprising administering to a subjecthaving type 2 diabetes mellitus, or at risk of developing type 2diabetes, an effective amount of an ALK7:ActRIIB antagonist (e.g., anALK7:ActRIIB heterodimer), or combination of such antagonists. In someembodiments, the subject has a body mass index BMI of 30 kg/m² orgreater (e.g., 30 to 39.9 kg/m²). In some embodiments, the subject has aBMI of at least 40 kg/m². In some embodiments, the subject has centralobesity (e.g., excess adiposity in the abdominal region, including bellyfat and/or visceral fat). In some embodiments, the subject has a WHR of0.85 or greater. In some embodiments, the subject has peripheral obesity(e.g., excess adiposity on the hips). In some embodiments, theALK7:ActRIIB antagonist treatment is an adjunct to diet and/or exercise.

Also provided is a method of treating, ameliorating or preventing adisease or condition associated with diabetes, comprising administeringto a subject having diabetes, an effective amount of an ALK7:ActRIIBantagonist (e.g., an ALK7:ActRIIB heterodimer), or combination of suchantagonists. In one embodiment, the treated or prevented disease orcondition is a member selected from the group consisting of:dyslipidemia, hyperlipidemia (total cholesterol level >240 mg/dL),hypercholesterolemia (e.g., total cholesterol level of >200 mg/dL, >220mg/dL, >240 mg/dL, >250 mg/dL, or >275 mg/dL), low HDL serum level(e.g., <40 mg/dL, <45 mg/dL, or <50 mg/dL), high LDL serum level (e.g.,≥100 mg/dL, ≥130 mg/dL, ≥160 mg/dL, or ≥190 mg/dL), andhypertriglyceridemia (e.g., a fasting TG level of ≥150 mg/dL, ≥175mg/dL, ≥200 mg/dL, ≥300 mg/dL, ≥400 mg/dL, or ≥499 mg/dL). In oneembodiment, the treated or prevented disease or condition iscardiovascular disease. In an additional embodiment, the treated orprevented disease or condition is hypertension (high blood pressure),myocardial infarction, peripheral artery disease, vasoregulatoindysfunction, or arteriosclerosis. In one embodiment, the treated orprevented disease or condition is liver disease. In another embodiment,the treated or prevented disease or condition is a member selected fromthe group: fatty liver disease, steatohepatitis, steatosis, and/orcirrhosis. In one embodiment, the treated or prevented disease orcondition is a member selected from the group consisting of: cataracts,obstructive sleep apnea, phlebitis, gout, osteoarthritis, gallbladderdisease, and high cholesterol. In certain instances, the ALK7:ActRIIBantagonist treatment is an adjunct to diet and/or exercise.

The disclosure also provides a method for improving the blood-lipidprofile in a subject, comprising administering to a subject in need ofsuch treatment an effective amount of an ALK7:ActRIIB antagonist (e.g.,an ALK7:ActRIIB heterodimer), or combination of such antagonists. Insome embodiments, the disclosure provides a method for reducing levelsof LDL cholesterol or increasing levels of HDL-cholesterol. In oneembodiment, the subject has dyslipidemia. In another embodiment, thesubject has elevated serum lipids (e.g., cholesterol(hypercholesterolemia) and/or triglycerides (e.g.,hypertriglyceridemia). In one embodiment the subject has an LDL-C ≥100mg/dL, ≥130 mg/dL, or ≥160 mg/dL). In one embodiment the subject has aTG ≥150 mg/dL, ≥160 mg/dL, ≥170 mg/dL). In one embodiment, the subjecthas elevated plasma insulin levels (hyperinsulinemia; e.g., fastinginsulin level of >20 ug/ml can exceed 100). In some embodiments, thesubject has type II diabetes.

According to one embodiment, the disclosure provides a method oftreating or preventing a metabolic disease or disorder or a conditionassociated with a metabolic disease or disorder, comprisingadministering an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combination of such antagonists, to a subject in needthereof. In one embodiment, the treated metabolic disease, disorder, orcondition is hyperglycemia (e.g., >130 mg/dL in the fasting state orfollowing glucose administration during an oral glucose tolerance test).In one embodiment, the treated metabolic disease, disorder, or conditionis a lipid metabolism disease, disorder, or condition. In oneembodiment, the treated metabolic disease, disorder, or condition isdislipidemia. In a further embodiment, the lipid metabolism disease,disorder, or condition is a member selected from: low HDL levels, highLDL levels, high triglyceride levels, hyperlipidemia, and a lipoproteinaberration. In one embodiment, the subject has a total cholesterol levelof >200 mg/dL, >220 mg/dL, >240 mg/dL, >250 mg/dL, or >275 mg/dL. In oneembodiment, the subject has a HDL serum level of <40 mg/dL, <45 mg/dL,or <50 mg/dL). In one embodiment, the subject has a LDL serum level ≥100mg/dL, ≥130 mg/dL, ≥160 mg/dL, or ≥190 mg/dL. In one embodiment, thesubject has fasting TG level of ≥150 mg/dL, ≥175 mg/dL, ≥200 mg/dL, ≥300mg/dL, ≥400 mg/dL, or ≥499 mg/dL. In one embodiment, the treatedmetabolic disease, disorder, or condition is a glucose metabolismdisease, disorder, or condition. In a further embodiment, the glucosemetabolism disease, disorder, or condition is a member selected from:glucose intolerance, insulin resistance, impaired glucose tolerance(IGT), impaired fasting glucose (IFG). In one embodiment, the treatedmetabolic disease, disorder, or condition is a member selected from thegroup consisting of: high uric acid levels, NAFLD, fatty liver, NASH,and polycystic ovarian syndrome. In one embodiment, the treated subjecthas hyperinsulinemia. In one embodiment, the treated subject is obese(e.g., the subject has abdominal obesity). In another embodiment, thetreated subject has type II diabetes.

Metabolic syndrome is a condition involving a set of disorders thatenhances the risk of heart disease. The major components of metabolicsyndrome are excess weight, the cardiovascular parameters (high bloodpressure, dyslipidemia, high levels of triglycerides and/or low levelsof HDL in the blood), atherosclerosis, diabetes, and/or insulinresistance. A subject having several of these components, i.e. metabolicsyndrome, is highly prone to heart disease, though each component is arisk factor. The disclosure also provides a method for treating orpreventing 1, 2, 3, or more of the above components of metabolicsyndrome, comprising administering to a subject in need of treatment aneffective amount of an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combination of such antagonists.

Additionally provided is a method of treating, preventing orameliorating a cardiovascular disease or condition, comprisingadministering an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combination of such antagonists, to a subject in needthereof. In one embodiment, the treated, prevented, or amelioratedcardiovascular disease or condition is atherosclerosis. In oneembodiment, the treated, prevented, or ameliorated cardiovasculardisease or condition is hypertension (e.g., blood pressure >130/80 mmHgor >140/90 mmHg, in a resting state. In one embodiment, thecardiovascular disease is atherosclerosis (coronary heart diseasedisease).

In one embodiment, the disclosure provides a method for treating and/orameliorating an inflammatory liver disease or condition that comprisesadministering an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combination of such antagonists, to a subject in needthereof. In one embodiment, the disease or condition is NAFLD. In afurther embodiment, the disease or condition is fatty liver. In afurther embodiment, the disease or condition is steatosis (e.g.,nonalcoholic steatohepatitis (NASH)). In a further embodiment, thedisease or condition is alcoholic fatty liver disease.

This disclosure also provides a method of improving glycemic control,comprising administering to a subject in need of treatment an effectiveamount of an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer). In one embodiment, the subject is administered has afasting blood sugar level of >130, >135, >140, >145, or >150 mg/dL. Inone embodiment, the subject is administered has a postprandial bloodsugar level of >180, >185, >190, >195, or >200 mg/dL 2 hours aftereating. In certain instances, the ALK7:ActRIIB antagonist treatment isan adjunct to diet and/or exercise. The administration can also reducebody weight or treat obesity. In certain instances, the subject has type2 diabetes mellitus. In certain instances, the subject has a BMI of 27to 40 kg/m2. In certain instances, the subject has a BMI of 30 to 39.9kg/m2. In certain instances, the subject has a BMI of at least 40. Incertain instances, the subject is overweight. In certain instances, thesubject is obese. An improvement in glycemic control can be assessedusing techniques known in the art such as a mixed-meal test.

The disclosure also provides compositions and methods for treating,preventing or ameliorating hyperglycemia or a condition associated withhyperglycemia in a subject comprising administering to a subject in needof such treatment an effective amount of an ALK7:ActRIIB antagonist(e.g., an ALK7:ActRIIB heterodimer). In one embodiment, the subject isadministered has a fasting blood sugar level of >130, >135, >140, >145,or >150 mg/dL. In one embodiment, the subject is administered has apostprandial blood sugar level of >180, >185, >190, >195, or >200 mg/dL2 hours after eating. In one embodiment, the result of the treatment,prevention or amelioration is a member selected from the groupconsisting of: a decrease in serum levels of glucose, a decrease inserum levels of triglycerides, a decrease in serum levels of insulin,and/or a decrease in serum levels of non-esterified fatty acids, ascompared to serum levels in the subject prior to treatment. In oneembodiment, the result of the treatment, prevention or amelioration isan increase in body temperature of about 0.4° C. to 1° C. as compared tobody temperature of the subject prior to treatment. In some embodiments,the ALK7:ActRIIB treatment also reduces body weight of the subject.

In another embodiment, the disclosure provides a method of decreasingplasma insulin levels in a subject, comprising administering aneffective amount of an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), to a subject in need of such treatment. In one embodiment,the subject has a fasting blood sugar level of >130, >135, >140, >145,or >150 mg/dL. In one embodiment, the subject has a postprandial bloodsugar level of >180, >185, >190, >195, or >200 mg/dL 2 hours aftereating. In one embodiment, the subject is overweight. In one embodiment,the subject is obese. In another embodiment, the subject has type 2diabetes.

The disclosure also provides compositions and methods for treating,preventing or ameliorating hyperglycemia or a condition associated withhyperglycemia in a subject comprising administering to a subject in needof such treatment an effective amount of an ALK7:ActRIIB antagonist(e.g., an ALK7:ActRIIB heterodimer). In one embodiment, the subject hasa fasting blood sugar level of >130, >135, >140, >145, or >150 mg/dL. Inone embodiment, the subject has a postprandial blood sugar levelof >180, >185, >190, >195, or >200 mg/dL 2 hours after eating. In oneembodiment, the result of the treatment, prevention or amelioration is amember selected from the group consisting of: a decrease in serum levelsof glucose, a decrease in serum levels of triglycerides, a decrease inserum levels of insulin, and/or a decrease in serum levels ofnon-esterified fatty acids, as compared to serum levels in the subjectprior to treatment. In one embodiment, the result of the treatment,prevention or amelioration is an increase in body temperature of about0.4° C. to 1° C. as compared to body temperature of the subject prior totreatment. In some embodiments, the ALK7:ActRIIB antagonist treatmentalso reduces body weight of the subject.

In another embodiment, the disclosure provides a method of decreasingplasma insulin levels in a subject, comprising administering aneffective amount of an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combination of such antagonists, to a subject in needof such treatment. In one embodiment, the subject has a fasting bloodsugar level of >130, >135, >140, >145, or >150 mg/dL. In one embodiment,the has a postprandial blood sugar level of >180, >185, >190, >195,or >200 mg/dL 2 hours after eating. In one embodiment, the subject isoverweight. In one embodiment, the subject is obese. In anotherembodiment, the subject has type 2 diabetes.

In another embodiment, the disclosure provides a method of treating,preventing, or ameliorating liver disease in a subject, comprisingadministering an effective amount of an ALK7:ActRIIB antagonist (e.g.,an ALK7:ActRIIB heterodimer), or combination of such antagonists, to asubject having a liver disease. In one embodiment, the subject hasinflammation of the liver. In one embodiment, the subject has NAFLD. Inon embodiment the subject has fatty liver. In another embodiment, thesubject has NASH. In on embodiment the subject has fatty liver. Inanother embodiment, the subject has alcoholic fatty liver disease. Inone embodiment, the treated, prevented or ameliorated liver disease isfibrosis, scarring, cirrhosis, or liver failure. In another embodiment,the treated, prevented or ameliorated liver disease is liver cancer. Inone embodiment, the subject is overweight. In another embodiment, thesubject is obese. In another embodiment, the subject has type 2diabetes.

In on embodiment, the disclosure provides a method of for increasinglipolysis in a cell (e.g., white or brown adipose cells or tissue)comprising administering effective amount of an ALK7:ActRIIB antagonist(e.g., an ALK7:ActRIIB heterodimer), or combination of such antagonists.In some embodiments the cell is contacted in vitro. In some embodimentsthe cell is contacted in vivo. In one embodiment, the method is carriedout in vivo, for example, in a mammalian subject (e.g., an animalmodel). In a further embodiment, the subject is a human. In someembodiments, the method leads to increased glycerol production. Infurther embodiments, the method leads to increased glycerol and/or freefatty acid in an adipocyte culture. In some embodiments, the methodleads to decreased triglyceride (TG) content in the cell or tissue. Insome embodiments, the method leads to a decreased plasma TG level in asubject.

In another embodiment, the disclosure provides a method of increasingadrenergic receptor-β (ADRB) signaling in a cell or tissue (e.g., whiteor brown adipose cells or tissue). The method comprises contacting acell or tissue with an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combination of such antagonists, in an amountsufficient to increase ADRB signaling. In some embodiments the cell ortissue is contacted in vitro. In some embodiments the cell or tissue iscontacted in vivo. In one embodiment, the method is carried out in vivo,for example, in a mammalian subject (e.g., an animal model). In afurther embodiment, the subject is a human. In some embodiments, themethod leads to increased glycerol production. In further embodiments,the method leads to increased glycerol and/or free fatty acid in anadipocyte culture. In some embodiments, the method leads to decreased TGcontent in the cell or tissue. In some embodiments, the method leads toa decreased plasma TG level in a subject. In some embodiments, themethod leads to an increased ADRB signaling in an adipocyte or adiposetissue during nutrient overload.

In another embodiment, the disclosure provides a method of decreasingperoxisome proliferator-activated receptor-gamma (PPAR gamma) signalingin a cell or tissue (e.g., white and/or brown adipose cell or tissue).The method includes contacting a cell or tissue with an ALK7:ActRIIBantagonist (e.g., an ALK7:ActRIIB heterodimer), or combination of suchantagonists, in an amount effective to decrease PPAR gamma activity. Insome embodiments the cells or tissue is contacted in vitro. In someembodiments the differentiated cells or tissue is contacted in vivo. Inone embodiment, the method is carried out in vivo, for example, in amammalian subject (e.g., an animal model). In a further embodiment, thesubject is a human. In some embodiments, the method leads to increasedglycerol production. In further embodiments, the method leads toincreased glycerol and/or free fatty acid in an adipocyte culture. Insome embodiments, the method leads to decreased TO content in the cellsor tissue. In some embodiments, the method leads to a decreased plasmaTG level in a subject.

In another embodiment, the disclosure provides a method of decreasinginsulin resistance in a cell or tissue (e.g., white and/or brown adiposecell or tissue). The method includes contacting a cell or tissue with anALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIB heterodimer), orcombination of such antagonists, in an amount effective to reduceinsulin resistance. In some embodiments the cell or tissue is contactedin vitro. In some embodiments the cell or tissue is contacted in vivo.In one embodiment, the method is carried out in vivo, for example, in amammalian subject (e.g., an animal model). In a further embodiment, thesubject is a human.

In another embodiment, the disclosure provides a method of increasingthe metabolic rate of a cell or tissue (e.g., white and/or brown adiposecell or tissue). The method includes contacting cell or tissue with anALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIB heterodimer), orcombination of such antagonists, in an amount effective to increasemetabolism of the cell or tissue. In some embodiments the cell or tissueis contacted in vitro. In some embodiments the cell or tissue iscontacted in vivo. In one embodiment, the method is carried out in vivo,for example, in a mammalian subject (e.g., an animal model). In afurther embodiment, the subject is a human.

The disclosure provides methods that comprise administering atherapeutically effective amount of an ALK7:ActRIIB antagonist (e.g., anALK7:ActRIIB heterodimer), or combination of such antagonists, alone orin combination with one or more additional therapies (e.g., one or moreadditional therapeutic agents and/or supportive) to a subject having, orat risk for developing, an ALK7-mediated disease and/or condition suchas, obesity (e.g., abdominal obesity); overweight; insulin resistance;metabolic syndrome and other metabolic diseases or conditions; a lipiddisorder such as, low HDL levels, high LDL levels, hyperlipidemia,hypertriglyceridemia or dyslipidemia; lipoprotein aberrations; decreasedtriglycerides; fatty liver disease; non-alcoholic fatty liver disease;hyperglycemia; impaired glucose tolerance (IGT); hyperinsulinemia; highcholesterol (e.g., high LDL levels and/or hypercholesterolemia);cardiovascular disease such as, heart disease including coronary heartdisease, congestive heart failure, atherosclerosis; arteriosclerosis,and/or hypertension; Syndrome X; vascular restenosis; neuropathy; and/orother disorders/conditions associated with one or more of the abovediseases or conditions, and/or with overweight (e.g., BMI of ≥25 kg/m²),or with too much body fat.

In additional embodiments, the disclosure provides methods of treatingand/or ameliorating cancer or a condition associated with cancer, thatcomprises administering an ALK7:ActRIIB antagonist (e.g., anALK7:ActRIIB heterodimer), or combination of such antagonists. In someembodiments, the subject has a cancer selected from the group consistingof melanoma, breast, colon, and endometrial, pancreatic, gastric, anduterine cancer. In some embodiments, the subject has myeloma (e.g.,multiple myeloma, plasmacytoma, localized myeloma, and extramedullarymyeloma). In some embodiments, the ALK7:ActRIIB antagonist isadministered to treat or prevent lymphatic metastasis, bloodstreammetastasis, tumor growth, or tumor invasion.

Fibrosis generally refers to an excessive deposition of both collagenfibers and extracellular matrix combined with a relative decrease ofcell number in an organ or tissue. While this process is an importantfeature of natural wound healing following injury, fibrosis can lead topathological damage in various tissue and organs including, for example,the lungs, kidneys, liver, bone, muscle, and skin. The role TGF-beta infibrosis has been extensively study. However, other TGF-beta superfamilyligands have also been implicated in fibrosis including, for example,activins (e.g., activin A and activin B) and GDF8 [Hedger et al (2013)Cytokine and Growth Factor Reviews 24:285-295; Hardy et al. (2015) 93:567-574; and Cantini et al. (2008) J Sex Med 5:1607-1622]. Therefore, insome embodiments, an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combinations of such antagonists, of the presentdisclosure can be used to treat fibrosis, particularlyfibrosis-associated disorders and conditions. For example, anALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIB heterodimer), orcombinations of such antagonists, may be used to treat or prevent one ormore of: pulmonary fibrosis, hypersensitivity pneumonitis, idiopathicfibrosis, tuberculosis, pneumonia, cystic fibrosis, asthma, chronicobstructive pulmonary disease (COPD), emphysema, renal (kidney)fibrosis, renal (kidney) failure, chronic renal (kidney) disease, bonefibrosis, myelofibrosis, rheumatoid arthritis, systemic lupuserythematosus, scleroderma, sarcoidosis, granulomatosis withpolyangiitis, Peyronie's disease, liver fibrosis, Wilson's disease,glycogen storage diseases (particularly types III, IV, IX, and X),iron-overload, Gaucher disease, Zellweger syndrome, nonalcoholic andalcoholic steatohepatitis, biliary cirrhosis, sclerosing cholangitis,Budd-Chiari syndrome, surgery-associated fibrosis, Crohn's disease,Duputren's contracture, mediastinal fibrosis, nephrogeneic fibrosis,retroperitoneal fibrosis, atrial fibrosis, endomyocardial fibrosis,pancreatic fibrosis.

In part, the present disclosure relate to the surprising discovery thatALK7:ActRIIB heteromultimers can bind to activin C. Therefore,ALK7:ActRIIB heteromultimers represent a new class of activin Cinhibitors that can be used to treat or prevent activin C-relateddisorders or conditions. In some embodiments, ALK7:ActRIIBheteromultimers may be used to inhibit activin C activity in a patientin need thereof. Overexpression of activin C has been implicated indisease and disorders associated with liver, testis, and prostate,particular with cancer in such tissues. See, e.g., Gold et al. Am JPathol (2009) 174(1): 184-195. In some embodiments, ALK7:ActRIIBheteromultimers may be used to increase male fertility, increase spermproduction, increase seminiferous tubule volume, decrease liverinflammation, treat liver (hepatic) cancer, treat testicular cancer,treat prostate cancer, decrease prostate inflammation, and/or treatprostate hypertrophy.

In some embodiments, ALK7:ActRIIB-associated conditions includeneuromuscular disorders (e.g., muscular dystrophy and muscle atrophy),congestive obstructive pulmonary disease (and muscle wasting associatedwith COPD), muscle wasting syndrome, sarcopenia, cachexia, adiposetissue disorders (e.g., obesity), type 2 diabetes (NIDDM, adult-onsetdiabetes), and bone degenerative disease (e.g., osteoporosis). Otherexemplary ALK7:ActRIIB-associated conditions include musculodegenerativeand neuromuscular disorders, tissue repair (e.g., wound healing),neurodegenerative diseases (e.g., amyotrophic lateral sclerosis), andimmunologic disorders (e.g., disorders related to abnormal proliferationor function of lymphocytes).

In certain embodiments, an ALK7:ActRIIB antagonist (e.g., anALK7:ActRIIB heterodimer), or combinations of such antagonists, of thedisclosure may be used as part of a treatment for a muscular dystrophy.The term “muscular dystrophy” refers to a group of degenerative musclediseases characterized by gradual weakening and deterioration ofskeletal muscles and sometimes the heart and respiratory muscles.Muscular dystrophies are genetic disorders characterized by progressivemuscle wasting and weakness that begin with microscopic changes in themuscle. As muscles degenerate over time, the person's muscle strengthdeclines. Exemplary muscular dystrophies that can be treated with aregimen including the subject TGF-beta superfamily heteromultimercomplexes include: Duchenne muscular dystrophy (DMD), Becker musculardystrophy (BMD), Emery-Dreifuss muscular dystrophy (EDMD), limb-girdlemuscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSHor FSHD) (also known as Landouzy-Dejerine), myotonic dystrophy (MMD;also known as Steinert's Disease), oculopharyngeal muscular dystrophy(OPMD), distal muscular dystrophy (DD), congenital muscular dystrophy(CMD).

Duchenne muscular dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. Beckermuscular dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is defective. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

BMD results from different mutations in the dystrophin gene. BMDpatients have some dystrophin, but it is either of insufficient quantityor poor quality. The presence of some dystrophin protects the muscles ofpatients with BMD from degenerating as severely or as quickly as thoseof patients with DMD.

Studies in animals indicate that inhibition of the GDF8 signalingpathway may effectively treat various aspects of disease in DMD and BMDpatients (Bogdanovich et al., 2002, Nature 420:418-421; Pistilli et al.,2011, Am J Pathol 178:1287-1297). Thus, ALK7:ActRIIB antagonists of thedisclosure may act as GDF8 inhibitors (antagonists), and constitute analternative means of blocking signaling by GDF8 and/or related TGFβsuperfamily ligands in vivo in DMD and BMD patients.

Similarly, ALK7:ActRIIB antagonists of the disclosure may provide aneffective means to increase muscle mass in other disease conditions thatare in need of muscle growth. For example, amyotrophic lateral sclerosis(ALS), also called Lou Gehrig's disease or motor neuron disease, is achronic, progressive, and incurable CNS disorder that attacks motorneurons, which are components of the central nervous system required forinitiation of skeletal muscle contraction. In ALS, motor neuronsdeteriorate and eventually die, and though a person's brain normallyremains fully functioning and alert, initiation of muscle contraction isblocked at the spinal level. Individuals who develop ALS are typicallybetween 40 and 70 years old, and the first motor neurons to degenerateare those innervating the arms or legs. Patients with ALS may havetrouble walking, may drop things, fall, slur their speech, and laugh orcry uncontrollably. As the disease progresses, muscles in the limbsbegin to atrophy from disuse. Muscle weakness becomes debilitating, andpatients eventually require a wheel chair or become confined to bed.Most ALS patients die from respiratory failure or from complications ofventilator assistance like pneumonia 3-5 years from disease onset.

Promotion of increased muscle mass by ALK7:ActRIIB antagonists mightalso benefit those suffering from muscle wasting diseases.Gonzalez-Cadavid et al. (supra) reported that GDF8 expression correlatesinversely with fat-free mass in humans and that increased expression ofthe GDF8 gene is associated with weight loss in men with AIDS wastingsyndrome. By inhibiting the function of GDF8 in AIDS patients, at leastcertain symptoms of AIDS may be alleviated, if not completelyeliminated, thus significantly improving quality of life in AIDSpatients.

Since loss of GDF8 function is also associated with fat loss withoutdiminution of nutrient intake (Zimmers et al., supra; McPherron and Lee,supra), the subject ALK7:ActRIIB antagonists may further be used as atherapeutic agent for slowing or preventing the development of obesityand type 2 diabetes.

Cancer anorexia-cachexia syndrome is among the most debilitating andlife-threatening aspects of cancer. This syndrome is a common feature ofmany types of cancer—present in approximately 80% of cancer patients atdeath—and is responsible not only for a poor quality of life and poorresponse to chemotherapy but also a shorter survival time than is foundin patients with comparable tumors but without weight loss. Cachexia istypically suspected in patients with cancer if an involuntary weightloss of greater than five percent of premorbid weight occurs within asix-month period. Associated with anorexia, wasting of fat and muscletissue, and psychological distress, cachexia arises from a complexinteraction between the cancer and the host. Cancer cachexia affectscytokine production, release of lipid-mobilizing andproteolysis-inducing factors, and alterations in intermediarymetabolism. Although anorexia is common, a decreased food intake aloneis unable to account for the changes in body composition seen in cancerpatients, and increasing nutrient intake is unable to reverse thewasting syndrome. Currently, there is no treatment to control or reversethe cachexic process. Since systemic overexpression of GDF8 in adultmice was found to induce profound muscle and fat loss analogous to thatseen in human cachexia syndromes (Zimmers et al., supra), the subjectALK7:ActRIIB antagonists may be beneficially used to prevent, treat, oralleviate the symptoms of the cachexia syndrome, where muscle growth isdesired. An example of a heteromeric complex useful for preventing,treating, or alleviating muscle loss as described above is anALK7:ActRIIB heterodimer.

In certain embodiments, an ALK7:ActRIIB antagonist (e.g., anALK7:ActRIIB heterodimer), or combinations of such antagonists, of thepresent disclosure may be used in methods of inducing bone and/orcartilage formation, preventing bone loss, increasing bonemineralization, preventing the demineralization of bone, and/orincreasing bone density. ALK7:ActRIIB antagonists may be useful inpatients who are diagnosed with subclinical low bone density, as aprotective measure against the development of osteoporosis.

In some embodiments, an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combinations of such antagonists, of the presentdisclosure may find medical utility in the healing of bone fractures andcartilage defects in humans and other animals. The subject methods andcompositions may also have prophylactic use in closed as well as openfracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agent is usefulfor repair of craniofacial defects that are congenital, trauma-induced,or caused by oncologic resection, and is also useful in cosmetic plasticsurgery. Further, methods and compositions of the invention may be usedin the treatment of periodontal disease and in other tooth repairprocesses. In certain cases, an ALK7:ActRIIB antagonist (e.g., anALK7:ActRIIB heterodimer), or combinations of such antagonists, mayprovide an environment to attract bone-forming cells, stimulate growthof bone-forming cells, or induce differentiation of progenitors ofbone-forming cells. An ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combinations of such antagonists, of the disclosure mayalso be useful in the treatment of osteoporosis. Further, ALK7:ActRIIBantagonists may be used in repair of cartilage defects andprevention/reversal of osteoarthritis. Examples of heteromeric complexesuseful for inducing bone formation, preventing bone loss, increasingbone mineralization, preventing the demineralization of bone, and/orincreasing bone density as described herein are ALK7:ActRIIBheterodimers.

Rosen et al. (ed) Primer on the Metabolic Bone Diseases and Disorders ofMineral Metabolism, 7^(th) ed. American Society for Bone and MineralResearch, Washington D.C. (incorporated herein by reference) provides anextensive discussion of bone disorders that may be subject to treatmentwith an ALK7:ActRIIB antagonist, or with combinations of suchantagonists. A partial listing is provided herein. Methods andcompositions of the invention can be applied to conditions characterizedby or causing bone loss, such as osteoporosis (including secondaryosteoporosis), hyperparathyroidism, chronic kidney disease mineral bonedisorder, sex hormone deprivation or ablation (e.g. androgen and/orestrogen), glucocorticoid treatment, rheumatoid arthritis, severe burns,hyperparathyroidism, hypercalcemia, hypocalcemia, hypophosphatemia,osteomalacia (including tumor-induced osteomalacia), hyperphosphatemia,vitamin D deficiency, hyperparathyroidism (including familialhyperparathyroidism) and pseudohypoparathyroidism, tumor metastases tobone, bone loss as a consequence of a tumor or chemotherapy, tumors ofthe bone and bone marrow (e.g., multiple myeloma), ischemic bonedisorders, periodontal disease and oral bone loss, Cushing's disease,Paget's disease, thyrotoxicosis, chronic diarrheal state ormalabsorption, renal tubular acidosis, or anorexia nervosa. Methods andcompositions of the invention may also be applied to conditionscharacterized by a failure of bone formation or healing, includingnon-union fractures, fractures that are otherwise slow to heal, fetaland neonatal bone dysplasias (e.g., hypocalcemia, hypercalcemia, calciumreceptor defects and vitamin D deficiency), osteonecrosis (includingosteonecrosis of the jaw) and osteogenesis imperfecta. Additionally, theanabolic effects will cause such antagonists to diminish bone painassociated with bone damage or erosion. As a consequence of theanti-resorptive effects, such antagonists may be useful to treatdisorders of abnormal bone formation, such as osteoblastic tumormetastases (e.g., associated with primary prostate or breast cancer),osteogenic osteosarcoma, osteopetrosis, progressive diaphysealdysplasia, endosteal hyperostosis, osteopoikilosis, and melorheostosis.Other disorders that may be treated include fibrous dysplasia andchondrodysplasias.

In another specific embodiment, the disclosure provides a therapeuticmethod and composition for repairing fractures and other conditionsrelated to cartilage and/or bone defects or periodontal diseases. Theinvention further provides therapeutic methods and compositions forwound healing and tissue repair. The types of wounds include, but arenot limited to, burns, incisions and ulcers. See, e.g., PCT PublicationNo. WO 84/01106. Such compositions comprise a therapeutically effectiveamount of at least one of the ALK7:ActRIIB antagonists of the disclosurein admixture with a pharmaceutically acceptable vehicle, carrier, ormatrix.

In some embodiments, an ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIBheterodimer), or combinations of such antagonists, of the disclosure canbe applied to conditions causing bone loss such as osteoporosis,hyperparathyroidism, Cushing's disease, thyrotoxicosis, chronicdiarrheal state or malabsorption, renal tubular acidosis, or anorexianervosa. It is commonly appreciated that being female, having a low bodyweight, and leading a sedentary lifestyle are risk factors forosteoporosis (loss of bone mineral density, leading to fracture risk).However, osteoporosis can also result from the long-term use of certainmedications. Osteoporosis resulting from drugs or another medicalcondition is known as secondary osteoporosis. In Cushing's disease, theexcess amount of cortisol produced by the body results in osteoporosisand fractures. The most common medications associated with secondaryosteoporosis are the corticosteroids, a class of drugs that act likecortisol, a hormone produced naturally by the adrenal glands. Althoughadequate levels of thyroid hormones are needed for the development ofthe skeleton, excess thyroid hormone can decrease bone mass over time.Antacids that contain aluminum can lead to bone loss when taken in highdoses by people with kidney problems, particularly those undergoingdialysis. Other medications that can cause secondary osteoporosisinclude phenytoin (Dilantin) and barbiturates that are used to preventseizures; methotrexate (Rheumatrex, Immunex, Folex PFS), a drug for someforms of arthritis, cancer, and immune disorders; cyclosporine(Sandimmune, Neoral), a drug used to treat some autoimmune diseases andto suppress the immune system in organ transplant patients; luteinizinghormone-releasing hormone agonists (Lupron, Zoladex), used to treatprostate cancer and endometriosis; heparin (Calciparine, Liquaemin), ananticlotting medication; and cholestyramine (Questran) and colestipol(Colestid), used to treat high cholesterol. Bone loss resulting fromcancer therapy is widely recognized and termed cancer therapy-inducedbone loss (CTIBL). Bone metastases can create cavities in the bone thatmay be corrected by treatment with An ALK7:ActRIIB antagonist (e.g., anALK7:ActRIIB heterodimer). Bone loss can also be caused by gum disease,a chronic infection in which bacteria located in gum recesses producetoxins and harmful enzymes.

In a further embodiment, the present disclosure provides methods andtherapeutic agents for treating diseases or disorders associated withabnormal or unwanted bone growth. For example, patients with thecongenital disorder fibrodysplasia ossificans progressiva (FOP) areafflicted by progressive ectopic bone growth in soft tissuesspontaneously or in response to tissue trauma, with a major impact onquality of life. Additionally, abnormal bone growth can occur after hipreplacement surgery and thus ruin the surgical outcome. This is a morecommon example of pathological bone growth and a situation in which thesubject methods and compositions may be therapeutically useful. The samemethods and compositions may also be useful for treating other forms ofabnormal bone growth (e.g., pathological growth of bone followingtrauma, burns or spinal cord injury), and for treating or preventing theundesirable conditions associated with the abnormal bone growth seen inconnection with metastatic prostate cancer or osteosarcoma.

In certain embodiments, an ALK7:ActRIIB antagonist (e.g., anALK7:ActRIIB heterodimer), or combinations of such antagonists, of thedisclosure may be used to promote bone formation in patients withcancer. Patients having certain tumors (e.g. prostate, breast, multiplemyeloma or any tumor causing hyperparathyroidism) are at high risk forbone loss due to tumor-induced bone loss, bone metastases, andtherapeutic agents. Such patients may be treated with a TGF-betasuperfamily heteromultimer complex, or a combination of complexes, evenin the absence of evidence of bone loss or bone metastases. Patients mayalso be monitored for evidence of bone loss or bone metastases, and maybe treated with an ALK7:ActRIIB antagonist in the event that indicatorssuggest an increased risk. Generally, DEXA scans are employed to assesschanges in bone density, while indicators of bone remodeling may be usedto assess the likelihood of bone metastases. Serum markers may bemonitored. Bone specific alkaline phosphatase (B SAP) is an enzyme thatis present in osteoblasts. Blood levels of BSAP are increased inpatients with bone metastasis and other conditions that result inincreased bone remodeling. Osteocalcin and procollagen peptides are alsoassociated with bone formation and bone metastases. Increases in BSAPhave been detected in patients with bone metastasis caused by prostatecancer, and to a lesser degree, in bone metastases from breast cancer.BMP7 levels are high in prostate cancer that has metastasized to bone,but not in bone metastases due to bladder, skin, liver, or lung cancer.Type I carboxy-terminal telopeptide (ICTP) is a crosslink found incollagen that is formed during to the resorption of bone. Since bone isconstantly being broken down and reformed, ICTP will be found throughoutthe body. However, at the site of bone metastasis, the level will besignificantly higher than in an area of normal bone. ICTP has been foundin high levels in bone metastasis due to prostate, lung, and breastcancer. Another collagen crosslink, Type I N-terminal telopeptide (NTx),is produced along with ICTP during bone turnover. The amount of NTx isincreased in bone metastasis caused by many different types of cancerincluding lung, prostate, and breast cancer. Also, the levels of NTxincrease with the progression of the bone metastasis. Therefore, thismarker can be used to both detect metastasis as well as measure theextent of the disease. Other markers of resorption include pyridinolineand deoxypyridinoline. Any increase in resorption markers or markers ofbone metastases indicate the need for therapy with an ALK7:ActRIIBantagonist in a patient.

An ALK7:ActRIIB antagonist (e.g., an ALK7:ActRIIB heterodimer), orcombinations of such antagonists, of the disclosure may be conjointlyadministered with other bone-active pharmaceutical agents. Conjointadministration may be accomplished by administration of a singleco-formulation, by simultaneous administration, or by administration atseparate times. ALK7:ActRIIB antagonists may be particularlyadvantageous if administered with other bone-active agents. A patientmay benefit from conjointly receiving an ALK7:ActRIIB antagonist complexand taking calcium supplements, vitamin D, appropriate exercise and/or,in some cases, other medication. Examples of other medications include,bisphosphonates (alendronate, ibandronate and risedronate), calcitonin,estrogens, parathyroid hormone and raloxifene. The bisphosphonates(alendronate, ibandronate and risedronate), calcitonin, estrogens andraloxifene affect the bone remodeling cycle and are classified asanti-resorptive medications. Bone remodeling consists of two distinctstages: bone resorption and bone formation. Anti-resorptive medicationsslow or stop the bone-resorbing portion of the bone-remodeling cycle butdo not slow the bone-forming portion of the cycle. As a result, newformation continues at a greater rate than bone resorption, and bonedensity may increase over time. Teriparatide, a form of parathyroidhormone, increases the rate of bone formation in the bone remodelingcycle. Alendronate is approved for both the prevention (5 mg per day or35 mg once a week) and treatment (10 mg per day or 70 mg once a week) ofpostmenopausal osteoporosis. Alendronate reduces bone loss, increasesbone density and reduces the risk of spine, wrist and hip fractures.Alendronate also is approved for treatment of glucocorticoid-inducedosteoporosis in men and women as a result of long-term use of thesemedications (i.e., prednisone and cortisone) and for the treatment ofosteoporosis in men. Alendronate plus vitamin D is approved for thetreatment of osteoporosis in postmenopausal women (70 mg once a weekplus vitamin D), and for treatment to improve bone mass in men withosteoporosis. Ibandronate is approved for the prevention and treatmentof postmenopausal osteoporosis. Taken as a once-a-month pill (150 mg),ibandronate should be taken on the same day each month. Ibandronatereduces bone loss, increases bone density and reduces the risk of spinefractures. Risedronate is approved for the prevention and treatment ofpostmenopausal osteoporosis. Taken daily (5 mg dose) or weekly (35 mgdose or 35 mg dose with calcium), risedronate slows bone loss, increasesbone density and reduces the risk of spine and non-spine fractures.Risedronate also is approved for use by men and women to prevent and/ortreat glucocorticoid-induced osteoporosis that results from long-termuse of these medications (i.e., prednisone or cortisone). Calcitonin isa naturally occurring hormone involved in calcium regulation and bonemetabolism. In women who are more than 5 years beyond menopause,calcitonin slows bone loss, increases spinal bone density, and mayrelieve the pain associated with bone fractures. Calcitonin reduces therisk of spinal fractures. Calcitonin is available as an injection(50-100 IU daily) or nasal spray (200 IU daily).

A patient may also benefit from conjointly receiving an ALK7:ActRIIBantagonist, or combinations of such antagonists, and additionalbone-active medications. Estrogen therapy (ET)/hormone therapy (HT) isapproved for the prevention of osteoporosis. ET has been shown to reducebone loss, increase bone density in both the spine and hip, and reducethe risk of hip and spinal fractures in postmenopausal women. ET isadministered most commonly in the form of a pill or skin patch thatdelivers a low dose of approximately 0.3 mg daily or a standard dose ofapproximately 0.625 mg daily and is effective even when started afterage 70. When estrogen is taken alone, it can increase a woman's risk ofdeveloping cancer of the uterine lining (endometrial cancer). Toeliminate this risk, healthcare providers prescribe the hormoneprogestin in combination with estrogen (hormone replacement therapy orHT) for those women who have an intact uterus. ET/HT relieves menopausesymptoms and has been shown to have a beneficial effect on bone health.Side effects may include vaginal bleeding, breast tenderness, mooddisturbances and gallbladder disease. Raloxifene, 60 mg a day, isapproved for the prevention and treatment of postmenopausalosteoporosis. It is from a class of drugs called Selective EstrogenReceptor Modulators (SERMs) that have been developed to provide thebeneficial effects of estrogens without their potential disadvantages.Raloxifene increases bone mass and reduces the risk of spine fractures.Data are not yet available to demonstrate that raloxifene can reduce therisk of hip and other non-spine fractures. Teriparatide, a form ofparathyroid hormone, is approved for the treatment of osteoporosis inpostmenopausal women and men who are at high risk for a fracture. Thismedication stimulates new bone formation and significantly increasesbone mineral density. In postmenopausal women, fracture reduction wasnoted in the spine, hip, foot, ribs and wrist. In men, fracturereduction was noted in the spine, but there were insufficient data toevaluate fracture reduction at other sites. Teriparatide isself-administered as a daily injection for up to 24 months.

As used herein, “in combination with”, “combinations of”, or “conjointadministration” refers to any form of administration such thatadditional therapies (e.g., second, third, fourth, etc.) are stilleffective in the body (e.g., multiple compounds are simultaneouslyeffective in the patient, which may include synergistic effects of thosecompounds). Effectiveness may not correlate to measurable concentrationof the agent in blood, serum, or plasma. For example, the differenttherapeutic compounds can be administered either in the same formulationor in separate formulations, either concomitantly or sequentially, andon different schedules. Thus, an individual who receives such treatmentcan benefit from a combined effect of different therapies. One or moreALK7:ActRIIB antagonists of the disclosure can be administeredconcurrently with, prior to, or subsequent to, one or more otheradditional agents or supportive therapies. In general, each therapeuticagent will be administered at a dose and/or on a time scheduledetermined for that particular agent. The particular combination toemploy in a regimen will take into account compatibility of theantagonist of the present disclosure with the therapy and/or thedesired.

5. Pharmaceutical Compositions

In certain aspects, ALK7:ActRIIB antagonists (e.g., ALK7:ActRIIBheteromultimers), or combinations of such antagonists, of the presentdisclosure can be administered alone or as a component of apharmaceutical formulation (also referred to as a therapeuticcomposition or pharmaceutical composition). A pharmaceutical formationrefers to a preparation which is in such form as to permit thebiological activity of an active ingredient (e.g., an agent of thepresent disclosure) contained therein to be effective and which containsno additional components which are unacceptably toxic to a subject towhich the formulation would be administered. The subject compounds maybe formulated for administration in any convenient way for use in humanor veterinary medicine. For example, one or more agents of the presentdisclosure may be formulated with a pharmaceutically acceptable carrier.A pharmaceutically acceptable carrier refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isgenerally nontoxic to a subject. A pharmaceutically acceptable carrierincludes, but is not limited to, a buffer, excipient, stabilizer, and/orpreservative. In general, pharmaceutical formulations for use in thepresent disclosure are in a pyrogen-free, physiologically-acceptableform when administered to a subject. Therapeutically useful agents otherthan those described herein, which may optionally be included in theformulation as described above, may be administered in combination withthe subject agents in the methods of the present disclosure.

In certain embodiments, compositions will be administered parenterally[e.g., by intravenous (I. V.) injection, intraarterial injection,intraosseous injection, intramuscular injection, intrathecal injection,subcutaneous injection, or intradermal injection]. Pharmaceuticalcompositions suitable for parenteral administration may comprise one ormore agents of the disclosure in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use. Injectable solutions or dispersions maycontain antioxidants, buffers, bacteriostats, suspending agents,thickening agents, or solutes which render the formulation isotonic withthe blood of the intended recipient. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalformulations of the present disclosure include water, ethanol, polyols(e.g., glycerol, propylene glycol, polyethylene glycol, etc.), vegetableoils (e.g., olive oil), injectable organic esters (e.g., ethyl oleate),and suitable mixtures thereof. Proper fluidity can be maintained, forexample, by the use of coating materials (e.g., lecithin), by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

In some embodiments, a therapeutic method of the present disclosureincludes administering the pharmaceutical composition systemically, orlocally, from an implant or device. Further, the pharmaceuticalcomposition may be encapsulated or injected in a form for delivery to atarget tissue site (e.g., bone marrow or muscle). In certainembodiments, compositions of the present disclosure may include a matrixcapable of delivering one or more of the agents of the presentdisclosure to a target tissue site (e.g., bone marrow or muscle),providing a structure for the developing tissue and optimally capable ofbeing resorbed into the body. For example, the matrix may provide slowrelease of one or more agents of the present disclosure. Such matricesmay be formed of materials presently in use for other implanted medicalapplications.

In some embodiments, pharmaceutical compositions will be administered tothe eye including, e.g., by topical administration, intraocular (e.g.,intravitreal) injection, or by implant or device. An intravitrealinjection can be injected, for example, through the pars plana, 3 mm to4 mm posterior to the limbus. Pharmaceutical compositions foradministration to the eye may formulated in a variety of ways including,for example, eye drops, ophthalmic solutions, ophthalmic suspensions,ophthalmic emulsions, intravitreal injections, sub-Tenon injections,ophthalmic bioerodible implant, and non-bioerodible ophthalmic insertsor depots.

The choice of matrix material may be based on one or more of:biocompatibility, biodegradability, mechanical properties, cosmeticappearance, and interface properties. The particular application of thesubject compositions will define the appropriate formulation. Potentialmatrices for the compositions may be biodegradable and chemicallydefined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylacticacid, and polyanhydrides. Other potential materials are biodegradableand biologically well-defined including, for example, bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenon-biodegradable and chemically defined including, for example,sintered hydroxyapatite, bioglass, aluminates, or other ceramics.Matrices may be comprised of combinations of any of the above mentionedtypes of material including, for example, polylactic acid andhydroxyapatite or collagen and tricalciumphosphate. The bioceramics maybe altered in composition (e.g., calcium-aluminate-phosphate) andprocessing to alter one or more of pore size, particle size, particleshape, and biodegradability.

In certain embodiments, pharmaceutical compositions of presentdisclosure can be administered topically. “Topical application” or“topically” means contact of the pharmaceutical composition with bodysurfaces including, for example, the skin, wound sites, and mucousmembranes. The topical pharmaceutical compositions can have variousapplication forms and typically comprises a drug-containing layer, whichis adapted to be placed near to or in direct contact with the tissueupon topically administering the composition. Pharmaceuticalcompositions suitable for topical administration may comprise one ormore one or more TGFβ superfamily type I and/or type II receptorpolypeptide complexes of the disclosure in combination formulated as aliquid, a gel, a cream, a lotion, an ointment, a foam, a paste, a putty,a semi-solid, or a solid. Compositions in the liquid, gel, cream,lotion, ointment, foam, paste, or putty form can be applied byspreading, spraying, smearing, dabbing or rolling the composition on thetarget tissue. The compositions also may be impregnated into steriledressings, transdermal patches, plasters, and bandages. Compositions ofthe putty, semi-solid or solid forms may be deformable. They may beelastic or non-elastic (e.g., flexible or rigid). In certain aspects,the composition forms part of a composite and can include fibers,particulates, or multiple layers with the same or differentcompositions.

Topical compositions in the liquid form may include pharmaceuticallyacceptable solutions, emulsions, microemulsions, and suspensions. Inaddition to the active ingredient(s), the liquid dosage form may containan inert diluent commonly used in the art including, for example, wateror other solvent, a solubilizing agent and/or emulsifier [e.g., ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, or 1,3-butylene glycol, anoil (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesameoil), glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fattyacid ester of sorbitan, and mixtures thereof].

Topical gel, cream, lotion, ointment, semi-solid or solid compositionsmay include one or more thickening agents, such as a polysaccharide,synthetic polymer or protein-based polymer. In one embodiment of theinvention, the gelling agent herein is one that is suitably nontoxic andgives the desired viscosity. The thickening agents may include polymers,copolymers, and monomers of: vinylpyrrolidones, methacrylamides,acrylamides N-vinylimidazoles, carboxy vinyls, vinyl esters, vinylethers, silicones, polyethyleneoxides, polyethyleneglycols,vinylalcohols, sodium acrylates, acrylates, maleic acids,NN-dimethylacrylamides, diacetone acrylamides, acrylamides, acryloylmorpholine, pluronic, collagens, polyacrylamides, polyacrylates,polyvinyl alcohols, polyvinylenes, polyvinyl silicates, polyacrylatessubstituted with a sugar (e.g., sucrose, glucose, glucosamines,galactose, trehalose, mannose, or lactose), acylamidopropane sulfonicacids, tetramethoxyorthosilicates, methyltrimethoxyorthosilicates,tetraalkoxyorthosilicates, trialkoxyorthosilicates, glycols, propyleneglycol, glycerine, polysaccharides, alginates, dextrans, cyclodextrin,celluloses, modified celluloses, oxidized celluloses, chitosans,chitins, guars, carrageenans, hyaluronic acids, inulin, starches,modified starches, agarose, methylcelluloses, plant gums, hylaronans,hydrogels, gelatins, glycosaminoglycans, carboxymethyl celluloses,hydroxyethyl celluloses, hydroxy propyl methyl celluloses, pectins,low-methoxy pectins, cross-linked dextrans, starch-acrylonitrile graftcopolymers, starch sodium polyacrylate, hydroxyethyl methacrylates,hydroxyl ethyl acrylates, polyvinylene, polyethylvinylethers, polymethylmethacrylates, polystyrenes, polyurethanes, polyalkanoates, polylacticacids, polylactates, poly(3-hydroxybutyrate), sulfonated hydrogels, AMPS(2-acrylamido-2-methyl-1-propanesulfonic acid), SEM(sulfoethylmethacrylate), SPM (sulfopropyl methacrylate), SPA(sulfopropyl acrylate),N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine,methacrylic acid amidopropyl-dimethyl ammonium sulfobetaine, SPI(itaconic acid-bis(1-propyl sulfonizacid-3) ester di-potassium salt),itaconic acids, AMBC (3-acrylamido-3-methylbutanoic acid),beta-carboxyethyl acrylate (acrylic acid dimers), and maleicanhydride-methylvinyl ether polymers, derivatives thereof, saltsthereof, acids thereof, and combinations thereof. In certainembodiments, pharmaceutical compositions of present disclosure can beadministered orally, for example, in the form of capsules, cachets,pills, tablets, lozenges (using a flavored basis such as sucrose andacacia or tragacanth), powders, granules, a solution or a suspension inan aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquidemulsion, or an elixir or syrup, or pastille (using an inert base, suchas gelatin and glycerin, or sucrose and acacia), and/or a mouth wash,each containing a predetermined amount of a compound of the presentdisclosure and optionally one or more other active ingredients. Acompound of the present disclosure and optionally one or more otheractive ingredients may also be administered as a bolus, electuary, orpaste.

In solid dosage forms for oral administration (e.g., capsules, tablets,pills, dragees, powders, and granules), one or more compounds of thepresent disclosure may be mixed with one or more pharmaceuticallyacceptable carriers including, for example, sodium citrate, dicalciumphosphate, a filler or extender (e.g., a starch, lactose, sucrose,glucose, mannitol, and silicic acid), a binder (e.g.carboxymethylcellulose, an alginate, gelatin, polyvinyl pyrrolidone,sucrose, and acacia), a humectant (e.g., glycerol), a disintegratingagent (e.g., agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, a silicate, and sodium carbonate), a solution retardingagent (e.g. paraffin), an absorption accelerator (e.g. a quaternaryammonium compound), a wetting agent (e.g., cetyl alcohol and glycerolmonostearate), an absorbent (e.g., kaolin and bentonite clay), alubricant (e.g., a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate), a coloring agent, andmixtures thereof. In the case of capsules, tablets, and pills, thepharmaceutical formulation (composition) may also comprise a bufferingagent. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using one or moreexcipients including, e.g., lactose or a milk sugar as well as a highmolecular-weight polyethylene glycol.

Liquid dosage forms for oral administration of the pharmaceuticalcomposition may include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and elixirs. In additionto the active ingredient(s), the liquid dosage form may contain an inertdiluent commonly used in the art including, for example, water or othersolvent, a solubilizing agent and/or emulsifier [e.g., ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, or 1,3-butylene glycol, an oil (e.g.,cottonseed, groundnut, corn, germ, olive, castor, and sesame oil),glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fatty acidester of sorbitan, and mixtures thereof]. Besides inert diluents, theoral formulation can also include an adjuvant including, for example, awetting agent, an emulsifying and suspending agent, a sweetening agent,a flavoring agent, a coloring agent, a perfuming agent, a preservativeagent, and combinations thereof.

Suspensions, in addition to the active compounds, may contain suspendingagents including, for example, an ethoxylated isostearyl alcohol,polyoxyethylene sorbitol, a sorbitan ester, microcrystalline cellulose,aluminum metahydroxide, bentonite, agar-agar, tragacanth, andcombinations thereof.

Prevention of the action and/or growth of microorganisms may be ensuredby the inclusion of various antibacterial and antifungal agentsincluding, for example, paraben, chlorobutanol, and phenol sorbic acid.

In certain embodiments, it may be desirable to include an isotonic agentincluding, for example, a sugar or sodium chloride into thecompositions. In addition, prolonged absorption of an injectablepharmaceutical form may be brought about by the inclusion of an agentthat delay absorption including, for example, aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the one or more of the agents of the present disclosure. In the caseof ALK7:ActRIIB antagonists that promote red blood cell formation,various factors may include, but are not limited to, the patient's redblood cell count, hemoglobin level, the desired target red blood cellcount, the patient's age, the patient's sex, the patient's diet, theseverity of any disease that may be contributing to a depressed redblood cell level, the time of administration, and other clinicalfactors. The addition of other known active agents to the finalcomposition may also affect the dosage. Progress can be monitored byperiodic assessment of one or more of red blood cell levels, hemoglobinlevels, reticulocyte levels, and other indicators of the hematopoieticprocess.

In certain embodiments, the present disclosure also provides genetherapy for the in vivo production of one or more of the agents of thepresent disclosure. Such therapy would achieve its therapeutic effect byintroduction of the agent sequences into cells or tissues having one ormore of the disorders as listed above. Delivery of the agent sequencescan be achieved, for example, by using a recombinant expression vectorsuch as a chimeric virus or a colloidal dispersion system. Preferredtherapeutic delivery of one or more of agent sequences of the disclosureis the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or an RNA virus(e.g., a retrovirus). The retroviral vector may be a derivative of amurine or avian retrovirus. Examples of retroviral vectors in which asingle foreign gene can be inserted include, but are not limited to:Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. Retroviral vectors can be made target-specific by attaching,for example, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing one or more of theagents of the present disclosure.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes (gag, pol, and env),by conventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for one or more of the agents of thepresent disclosure is a colloidal dispersion system. Colloidaldispersion systems include, for example, macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Incertain embodiments, the preferred colloidal system of this disclosureis a liposome. Liposomes are artificial membrane vesicles which areuseful as delivery vehicles in vitro and in vivo. RNA, DNA, and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form [Fraley, et al. (1981) TrendsBiochem. Sci., 6:77]. Methods for efficient gene transfer using aliposome vehicle are known in the art [Mannino, et al. (1988)Biotechniques, 6:682, 1988].

The composition of the liposome is usually a combination ofphospholipids, which may include a steroid (e.g. cholesterol). Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations. Other phospholipids or other lipidsmay also be used including, for example a phosphatidyl compound (e.g.,phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, a sphingolipid, a cerebroside, and aganglioside), egg phosphatidylcholine, dipalmitoylphosphatidylcholine,and distearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1. Generation of an ActRIIB-Fc:ALK7-Fc Heterodimer

Applicants constructed a soluble ActRIIB-Fc:ALK7-Fc heteromeric complexcomprising the extracellular domains of human ActRIIB and human ALK7,which are each fused to an Fc domain with a linker positioned betweenthe extracellular domain and the Fc domain. The individual constructsare referred to as ActRIIB-Fc and ALK7-Fc, respectively.

A methodology for promoting formation of ActRIIB-Fc:ALK7-Fc heteromericcomplexes, as opposed to the ActRIIB-Fc or ALK7-Fc homodimericcomplexes, is to introduce alterations in the amino acid sequence of theFc domains to guide the formation of asymmetric heteromeric complexes.Many different approaches to making asymmetric interaction pairs usingFc domains are described in this disclosure.

In one approach, illustrated in the ActRIIB-Fc and ALK7-Fc polypeptidesequences disclosed below, respectively, one Fc domain is altered tointroduce cationic amino acids at the interaction face, while the otherFc domain is altered to introduce anionic amino acids at the interactionface. The ActRIIB-Fc fusion polypeptide and ALK7-Fc fusion polypeptideeach employ the tissue plasminogen activator (TPA) leader:MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 70).

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 71) is shown below:

(SEQ ID NO: 71) 1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE 101ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC 151PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV 201DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP 251APIEKTISKA KGQPREPQVY TLPPSRKEMT KNQVSLTCLV KGFYPSDIAV 301EWESNGQPEN NYKTTPPVLK SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH 351EALHNHYTQK SLSLSPGK

The leader (signal) sequence and linker are underlined. To promoteformation of the ActRIIB-Fc:ALK7-Fc heterodimer rather than either ofthe possible homodimeric complexes, two amino acid substitutions(replacing acidic amino acids with lysine) can be introduced into the Fcdomain of the ActRIIB fusion protein as indicated by double underlineabove. The amino acid sequence of SEQ ID NO: 71 may optionally beprovided with lysine (K) removed from the C-terminus.

This ActRIIB-Fc fusion protein is encoded by the following nucleic acidsequence (SEQ ID NO: 72):

(SEQ ID NO: 72) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC  51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG 101 AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC  151GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC  201CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT  251GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG  301GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA  351GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC  401CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC  451CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA  501ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG  551TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG  601GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA  651CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT  701GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA  751GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC  801ACAGGTGTAC ACCCTGCCCC CATCCCGGAA GGAGATGACC AAGAACCAGG  851TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG  901GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC  951CGTGCTGAAG TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG  1001ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT  1051GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG  1101 TAAA

The mature ActRIIB-Fc fusion polypeptide (SEQ ID NO: 73) is as follows,and may optionally be provided with lysine removed from the C-terminus.

(SEQ ID NO: 73) 1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHCYASWRNSSGT  51 IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA 101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS  151RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS  201VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS  251RKEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLKSDGSF  301FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 

The complementary form of ALK7-Fc fusion protein (SEQ ID NO: 74) is asfollows:

(SEQ ID NO: 74) 1 MDAMKRGLCC VLLLCGAVFV SPGAGLKCVC LLCDSSNFTCQTEGACWASV  51 MLTNGKEQVI KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLP 101 TASPNAPKLG PMETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR  151TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV  201LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR  251EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYDTTP PVLDSDGSFF  301LYSDLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G 

The signal sequence and linker sequence are underlined. To promoteformation of the ActRIIB-Fc:ALK7-Fc heterodimer rather than either ofthe possible homodimeric complexes, two amino acid substitutions(replacing lysines with aspartic acids) can be introduced into the Fcdomain of the fusion protein as indicated by double underline above. Theamino acid sequence of SEQ ID NO: 74 may optionally be provided with alysine added at the C-terminus.

This ALK7-Fc fusion protein is encoded by the following nucleic acid(SEQ ID NO: 75):

(SEQ ID NO: 75) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGCTGTGTGGAGC  51 AGTCTTCGTT TCGCCCGGCG CCGGACTGAA GTGTGTATGT CTTTTGTGTG 101 ATTCTTCAAA CTTTACCTGC CAAACAGAAG GAGCATGTTG GGCATCAGTC  151ATGCTAACCA ATGGAAAAGA GCAGGTGATC AAATCCTGTG TCTCCCTTCC  201AGAACTGAAT GCTCAAGTCT TCTGTCATAG TTCCAACAAT GTTACCAAAA  251CCGAATGCTG CTTCACAGAT TTTTGCAACA ACATAACACT GCACCTTCCA  301ACAGCATCAC CAAATGCCCC AAAACTTGGA CCCATGGAGA CCGGTGGTGG  351AACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT  401CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG  451ACCCCTGAGG TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA  501GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA  551CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC  601CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA  651GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG  701CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG  751GAGGAGATGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT  801CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA  851ACAACTACGA CACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC  901CTCTATAGCG ACCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT  951CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA  1001AGAGCCTCTC CCTGTCTCCG GGT

The mature ALK7-Fc fusion protein sequence (SEQ ID NO: 76) is expectedto be as follows and may optionally be provided with a lysine added atthe C-terminus.

(SEQ ID NO: 76) 1 GLKCVCLLCD SSNFTCQTEG ACWASVMLTN GKEQVIKSCVSLPELNAQVF  51 CHSSNNVTKT ECCFTDFCNN ITLHLPTASP NAPKLGPMET GGGTHTCPPC 101 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV  151DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP  201APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV  251EWESNGQPEN NYDTTPPVLD SDGSFFLYSD LTVDKSRWQQ GNVFSCSVMH  301EALHNHYTQK SLSLSPG 

The ActRIIB-Fc and ALK7-Fc fusion proteins of SEQ ID NO: 71 and SEQ IDNO: 74, respectively, may be co-expressed and purified from a CHO cellline to give rise to a heteromeric complex comprisingActRIIB-Fc:ALK7-Fc.

In another approach to promote the formation of heteromultimer complexesusing asymmetric Fc fusion proteins, the Fc domains are altered tointroduce complementary hydrophobic interactions and an additionalintermolecular disulfide bond as illustrated in the ActRIIB-Fc andALK7-Fc polypeptide sequences of disclosed below.

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 77) is shown below:

(SEQ ID NO: 77) 1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNANWELERTNQS  51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE 101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC  151PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV  201DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP  251APIEKTISKA KGQPREPQVY TLPPCREEMT KNQVSLWCLV KGFYPSDIAV  301EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH  351EALHNHYTQK SLSLSPGK 

The leader sequence and linker are underlined. To promote formation ofthe ActRIIB-Fc:ALK7-Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing a serinewith a cysteine and a threonine with a tryptophan) can be introducedinto the Fc domain of the fusion protein as indicated by doubleunderline above. The amino acid sequence of SEQ ID NO: 77 may optionallybe provided with lysine removed from the C-terminus.

The mature ActRIIB-Fc fusion polypeptide (SEQ ID NO: 78) is as followsand may optionally be provided with lysine removed from the C-terminus.

(SEQ ID NO: 78) 1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHCYASWRNSSGT  51 IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA 101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS  151RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS  201VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC  251REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF  301FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 

The complementary form of ALK7-Fc fusion polypeptide (SEQ ID NO: 79) isas follows:

(SEQ ID NO: 79) 1 MDAMKRGLCC VLLLCGAVFV SPGAGLKCVC LLCDSSNFTCQTEGACWASV  51 MLTNGKEQVI KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLP 101 TASPNAPKLG PMETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR  151TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV  201LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVCTLPPSR  251EEMTKNQVSL SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF  301LVSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 

The leader sequence and linker sequence are underlined. To guideheterodimer formation with the ActRIIB-Fc fusion polypeptide of SEQ IDNOs 76 and 78 above, four amino acid substitutions can be introducedinto the Fc domain of the ALK7 fusion polypeptide as indicated by doubleunderline above. The amino acid sequence of SEQ ID NO: 79 may optionallybe provided with the lysine removed from the C-terminus.

The mature ALK7-Fc fusion protein sequence (SEQ ID NO: 80) is expectedto be as follows and may optionally be provided with the lysine removedfrom the C-terminus.

(SEQ ID NO: 80) 1 GLKCVCLLCD SSNFTCQTEG ACWASVMLTN GKEQVIKSCVSLPELNAQVF  51 CHSSNNVTKT ECCFTDFCNN ITLHLPTASP NAPKLGPMET GGGTHTCPPC 101 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV  151DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP  201APIEKTISKA KGQPREPQVC TLPPSREEMT KNQVSLSCAV KGFYPSDIAV  251EWESNGQPEN NYKTTPPVLD SDGSFFLVSK LTVDKSRWQQ GNVFSCSVMH  301EALHNHYTQK SLSLSPGK 

The ActRIIB-Fc and ALK7-Fc proteins of SEQ ID NO: 77 and SEQ ID NO: 79,respectively, may be co-expressed and purified from a CHO cell line, togive rise to a heteromeric complex comprising ActRIIB-Fc:ALK7-Fc.

Purification of various ActRIIB-Fc:ALK7-Fc complexes could be achievedby a series of column chromatography steps, including, for example,three or more of the following, in any order: protein A chromatography,Q sepharose chromatography, phenylsepharose chromatography, sizeexclusion chromatography, and cation exchange chromatography. Thepurification could be completed with viral filtration and bufferexchange.

Example 2. Ligand Binding Profile of ActRIIB-Fc:ALK7-Fc HeterodimerCompared to ActRIIB-Fc Homodimer and ALK7-Fc Homodimer

A Biacore™-based binding assay was used to compare ligand bindingselectivity of the ActRIIB-Fc:ALK7-Fc heterodimeric complex describedabove with that of ActRIIB-Fc and ALK7-Fc homodimeric complexes. TheActRIIB-Fc:ALK7-Fc heterodimer, ActRIIB-Fc homodimer, and ALK7-Fchomodimer were independently captured onto the system using an anti-Fcantibody. Ligands were injected and allowed to flow over the capturedreceptor protein. Results are summarized in the table below, in whichligand off-rates (k_(d)) most indicative of effective ligand traps aredenoted in bold.

Ligand binding profile of ActRIIB-Fc:ALK7-Fc heterodimer compared toActRIIB-Fc homodimer and ALK7-Fc homodimer ActRIM-Fc ALK7-FcActRIIB-Fc:ALK7-Fc homodimer homodimer heterodimer k_(a) k_(d) K_(D)k_(a) k_(d) K_(D) k_(a) k_(d) K_(D) Ligand (1/Ms) (1/s) (pM) (1/Ms)(1/s) (pM) (1/Ms) (1/s) (pM) activin A 1.3 × 1.4 × 11 No binding 4.4 ×1.9 × 43 10⁷ 10 ⁻⁴ 10⁷ 10⁻³ activin B 1.5 × 1.6 × 8 No binding 1.2 × 2.0× 17 10⁷ 10 ⁻⁴ 10⁷ 10 ⁻⁴ activin C No binding No binding 3.5 × 2.4 ×6900 10⁵ 10⁻³ activin AC 2.0 × 3.1 × 160 No binding 2.6 × 5.7 × 220 10⁷10⁻³ 10⁶ 10 ⁻⁴ BMP5 2.6 × 7.5 × 2900 No binding 1.5 × 85 × 57000 10⁷10⁻² 10⁵ 10⁻³ BMP6 2.4 × 3.9 × 160 No binding 1.2 × 6.3 × 5300 10⁷ 10⁻³10⁶ 10⁻³ BMP9 1.2 × 1.2 × 10 No binding Transient* >1400 10⁸ 10⁻³ BMP105.9 × 1.5 × 25 No binding 1.5 × 2.8 × 190 10⁶ 10 ⁻⁴ 10⁷ 10⁻³ GDF3 1.4 ×2.2 × 1500 No binding 2.3 × 1.0 × 4500 10⁶ 10⁻³ 10⁶ 10⁻² GDF8 3.5 × 2.4× 69 No binding 3.7 × 1.0 × 270 10⁶ 10 ⁻⁴ 10⁶ 10⁻³ GDF11 9.6 × 1.5 × 2No binding 9.5 × 7.5 × 8 10⁷ 10 ⁻⁴ 10⁷ 10 ⁻⁴ *Indeterminate due totransient nature of interaction

 Not tested

These comparative binding data demonstrate that the ActRIIB-Fc:ALK7-Fcheterodimer has an altered binding profile/selectivity relative toeither the ActRIIB-Fc homodimer or ALK7-Fc homodimer. Interestingly,four of the five ligands with the strongest binding to ActRIIB-Fchomodimer (activin A, BMP10, GDF8, and GDF11) exhibit reduced binding tothe ActRIIB-Fc:ALK7-Fc heterodimer, the exception being activin B whichretains tight binding to the heterodimer. Similarly, three of the fourligands with intermediate binding to ActRIIB-Fc homodimer (GDF3, BMP6,and particularly BMP9) exhibit reduced binding to the ActRIIB-Fc:ALK7-Fcheterodimer, whereas binding to activin AC is increased to become thesecond strongest ligand interaction with the heterodimer overall.Finally, activin C and BMP5 unexpectedly bind the ActRIIB-Fc:ALK7heterodimer with intermediate strength despite no binding (activin C) orweak binding (BMP5) to ActRIIB-Fc homodimer. The net result is that theActRIIB-Fc:ALK7-Fc heterodimer possesses a ligand-binding profiledistinctly different from that of either ActRIIB-Fc homodimer or ALK7-Fchomodimer, which binds none of the foregoing ligands. See FIG. 6 .

These results therefore demonstrate that the ActRIIB-Fc:ALK7-Fcheterodimer is a more selective antagonist of activin B and activin ACcompared to ActRIIB-Fc homodimer. Moreover, ActRIIB-Fc:ALK7-Fcheterodimer exhibits the unusual property of robust binding to activinC. Accordingly, an ActRIIB-Fc:ALK7-Fc heterodimer will be more usefulthan an ActRIIB-Fc homodimer in certain applications where suchselective antagonism is advantageous. Examples include therapeuticapplications where it is desirable to retain antagonism of activin B oractivin AC but decrease antagonism of one or more of activin A, GDF3,GDF8, GDF11, BMP9, or BMP10. Also included are therapeutic, diagnostic,or analytic applications in which it is desirable to antagonize activinC or, based on the similarity between activin C and activin E, activinE.

Example 3. ALK7:ActRIIB Heteromultimer Treatment Suppresses KidneyFibrosis and Inflammation and Reduces Kidney Injury

The effects of the ALK7-Fc:ActRIIB-Fc heterodimer described in Example 2on kidney disease was assessed in a mouse unilateral ureteralobstruction model. See, e.g., Klahr and Morrissey (2002) Am J PhysiolRenal Physiol 283: F861-F875.

Twenty-four C57BL/6 male mice 12 weeks of age underwent left unilateralureteral ligation twice at the level of the lower pole of kidney. After3 days, eight mice were euthanized and kidneys from individual animalswere harvested to assess kidney injury. The remaining mice wererandomized into two groups: i) eight mice were injected subcutaneouslywith the ALK7-Fc:ActRIIB-Fc heterodimer at a dose of 10 mg/kg at day 3,day 7, day 10, and day 14 after surgery and a ii) eight mice wereinjected subcutaneously with vehicle control, phosphate buffered saline(PBS), at day 3, day 7, day 10, and day 14 after surgery. Both groupswere sacrificed at day 17 in accordance with the relevant Animal CareGuidelines. Half kidneys from individual animals were collected forhistology analysis (H&E, and Masson's Trichrome stain), from both theUUO kidney and contralateral kidney, and 1/4 kidneys were used for RNAextraction (RNeasy Midi Kit, Qiagen, IL).

Gene expression analysis on UUO kidney samples was performed to assesslevels of various genes. QRT-PCR was performed on a CFX Connect™Real-time PCR detection system (Bio-Rad, CA) to evaluate the expressionof various fibrotic genes (Col1a1, Col3a1, Fibronectin, PAI-1, CTGF, anda-SMA), inflammatory genes (Tnfa, and MCP1), cytokines (TGFβ1, TGFβ2,TGFβ3, and activin A), kidney injury genes (NGAL), Hypoxia-induciblefactor 1-alpha (HIF1a), and activin A receptor (ActRIIA). See FIG. 13 .Treatment of mice with ALK7-Fc:ActRIIB-Fc heterodimer significantlysuppressed the expression of fibrotic and inflammatory genes, inhibitedthe upregulation of TGFβ 1/2/3, activin A, and ActRIIa, and reducedkidney injury. Histology data confirmed that ALK7-Fc:ActRIIB-Fcheterodimer treatment significantly inhibited kidney fibrosis andreduced kidney injury in the UUO model.

Together, these data demonstrate that ALK7:ActRIIB heteromultimertreatment suppresses kidney fibrosis and inflammation and reduces kidneyinjury. Moreover, these data indicate that other ALK7:ActRIIBantagonists may be useful in the treatment or preventing of kidneydisease including, for example, antagonists of ALK7 and/orActRIIB-binding ligands (e.g., ligand antibodies and other ligand trapssuch as follistatin, Cerberus and Lefty), antagonists of ALK7 and/orActRIIB receptors, antagonists of ALK7 and/or ActRIIB downstreamsignaling mediators (e.g., Smads), and antagonists of TGFβ superfamilyco-receptors (e.g., antagonists of Crypto or Cryptic).

Example 4. ALK7:ActRIIB Heteromultimer Increases Lipolysis in Adipocytes

Lipolysis is the hydrolysis of triglycerides within the cell intoglycerol and free fatty acids. The glycerol and free fatty acids arethen released into the bloodstream or culture media. While lipolysisoccurs in essentially all cells, it is most abundant in white and brownadipocytes. ALK7 signaling is thought to suppress lipolysis and toconsequently lead to fat accumulation in adipocytes and adipose tissue.Accordingly, the effects of the ALK7-Fc:ActRIIB-Fc heterodimer describedin Example 2 on lipolysis in adipocytes was assessed.

Specifically, 3T3-L1 cells (supplied by ATCC; ATCC® CL-173™) were grownin Dulbecco's Modified Eagle Medium (ATCC; ATCC® 30-2002™) containing10% Bovine Serum (Life Technologies; 16170-060) until reachingconfluency. To induce differentiation, at 2 days post-confluency mediumwas replaced by fresh Dulbecco's Modified Eagle Medium (ATCC; ATCC®30-2002™) containing 10% fetal Bovine serum (Life Technologies;10082147), dexamethasone (Sigma, D8893), IBMX (Sigma, 17018) and insulin(Sigma, 10516) for 2 weeks. Accumulation of lipid droplets on the cells,as determined by microscopy, was used to confirm a completedifferentiation into mature adipocyte cells. Adipocytes were treatedovernight with vehicle (PBS), activin B (50 ng/ml) or co-treated withactivin B (50 ng/ml) and ALK7-Fc:ActRIIB-Fc heterodimer (5 μg/ml). Cellswere washed two times with PBS and incubated with lipolysis assay buffer(supplied by Abcam; ab185433). Lipolysis assay buffer was collectedafter 3 hours and glycerol levels were measured according tomanufacturer's instruction (Abcam; ab185433). It was determined that theALK7-Fc:ActRIIB-Fc heterodimer significantly increased lipolysisactivity by 44.36% in this cell-based assay.

Accordingly, these data demonstrate that ALK7-Fc:ActRIIB-Fc heterodimercan be used to antagonize ALK7-mediated suppression of lipolysis andthereby increase fatty acid breakdown in adipocytes. Moreover, thesedata indicate that ALK7-Fc:ActRIIB-Fc heterodimers as well as otherALK7:ActRIIB antagonists, such as those disclosed herein, may be used totreat a variety of disorder or conditions associated with low lipolysisactivity and/or excessive fatty acid accumulation in cells, particularlyadipocytes, including for example, obesity, diabetes, insulinresistance; metabolic syndrome, fatty liver disease and other metabolicdiseases or conditions, particularly those associated with excess fatlevels.

We claim:
 1. A method of treating obesity in a subject in need thereof,comprising administering to the subject a soluble recombinantheteromultimer comprising an ALK7 polypeptide and an ActRIIBpolypeptide; wherein the ALK7 polypeptide comprises an amino acidsequence that is at least 90% identical to amino acids 28-92 of SEQ IDNO: 9 and the ActRIIB polypeptide comprises an amino acid sequence thatis at least 90% identical to amino acids 29-109 of SEQ ID NO: 1; whereinthe ALK7 and/or ActRIIB polypeptide is a fusion protein furthercomprising a heterologous domain; and wherein the heteromultimer bindsto one or more ligands selected from the group consisting of: activin B,activin C, and activin AC.
 2. The method of claim 1, wherein theheterologous domain comprises an Fc immunoglobulin domain.
 3. The methodof claim 2, wherein the Fc immunoglobulin domain comprises one or moreamino acid modifications that promotes heterodimer formation.
 4. Themethod of claim 2, wherein the immunoglobulin Fc domain comprises one ormore amino acid modifications that inhibit homodimer formation.
 5. Themethod of claim 2, wherein the heterologous domain comprises an Fcimmunoglobulin domain from an IgG immunoglobulin.
 6. The method of claim1, wherein the fusion protein further comprises a linker domainpositioned between the ALK7 domain and the heterologous domain and/or alinker domain positioned between the ActRIIB domain and the heterologousdomain.
 7. The method of claim 1, wherein the ALK7 polypeptide and/orActRIIB polypeptide comprises one or more modified amino acid residuesselected from the group consisting of: a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, and an amino acid conjugated to a lipidmoiety.
 8. The method of claim 1, wherein the heteromultimer is anALK7:ActRIIB heterodimer.
 9. The method of claim 1, wherein the ALK7polypeptide comprises an amino acid sequence that is at least 95%identical to amino acids 28-92 of SEQ ID NO: 9, and wherein the ActRIIBpolypeptide comprises an amino acid sequence that is at least 95%identical to amino acids 29-109 of SEQ ID NO:
 1. 10. The method of claim9, wherein the heterologous domain comprises an Fc immunoglobulindomain, wherein the Fc immunoglobulin domain is an IgG1 immunoglobulindomain.
 11. The method of claim 9, wherein the fusion protein furthercomprises a linker domain positioned between the ALK7 domain and theheterologous domain and/or a linker domain positioned between theActRIIB domain and the heterologous domain.
 12. The method of claim 1,wherein the ALK7 polypeptide comprises the amino acid sequencecorresponding to amino acids 28-92 of SEQ ID NO: 9, and wherein theActRIIB polypeptide comprises the amino acid sequence corresponding toamino acids 29-109 of SEQ ID NO:
 1. 13. The method of claim 12, whereinthe heterologous domain comprises an Fc immunoglobulin domain, whereinthe Fc immunoglobulin domain is an IgG1 immunoglobulin domain.
 14. Themethod of claim 12, wherein the fusion protein further comprises alinker domain positioned between the ALK7 domain and the heterologousdomain and/or a linker domain positioned between the ActRIIB domain andthe heterologous domain.
 15. The method of claim 1, wherein the ALK7polypeptide comprises an amino acid sequence that is at least 90%identical to the amino acid sequence of SEQ ID NO: 46, and wherein theActRIIB polypeptide comprises an amino acid sequence that is at least90% identical to the amino acid sequence of SEQ ID NO:
 2. 16. The methodof claim 1, wherein the ALK7 polypeptide comprises an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO: 46, and wherein the ActRIIB polypeptide comprises an aminoacid sequence that is at least 95% identical to the amino acid sequenceof SEQ ID NO:
 2. 17. The method of claim 16, wherein the heterologousdomain comprises an Fc immunoglobulin domain, wherein the Fcimmunoglobulin domain is an IgG1 immunoglobulin domain.
 18. The methodof claim 16, wherein the fusion protein further comprises a linkerdomain positioned between the ALK7 domain and the heterologous domainand/or a linker domain positioned between the ActRIIB domain and theheterologous domain.
 19. The method of claim 1, wherein the ALK7polypeptide comprises the amino acid sequence of SEQ ID NO: 46, andwherein the ActRIIB polypeptide comprises the amino acid sequence of SEQID NO:
 2. 20. The method of claim 19, wherein the heterologous domaincomprises an Fc immunoglobulin domain, wherein the Fc immunoglobulindomain is an IgG1 immunoglobulin domain.
 21. The method of claim 19,wherein the fusion protein further comprises a linker domain positionedbetween the ALK7 domain and the heterologous domain and/or a linkerdomain positioned between the ActRIIB domain and the heterologousdomain.